Method of treating or preventing disease associated with deposit of amyloid proteins (versions)

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology, namely to the application of a conjugate of an immunogenic peptide and can be used in medicine. The said conjugate, containing 1÷93 of immunogenic peptides Aβ(35-42) (SEQ ID NO: 2), or 1÷93 of immunogenic peptides Aβ(33-42) (SEQ ID NO: 3), or 1÷93 of immunogenic peptides Aβ(33-40) (SEQ ID NO: 4), connected with a native albumin by means of a linker region, containing cysteine and a bifunctional linker, on condition that the linker region is connected by means of cysteine to the N-end of not more than one immunogenic peptide, can be used for the application in treatment or prevention of a disease, associated with the deposit of amyloid proteins. The invention also relates to the application of a pharmaceutical composition, containing the said conjugate, for the treatment or prevention of the disease, associated with the deposit of the amyloid proteins.

EFFECT: invention makes it possible to efficiently reduce levels of the amyloid peptide with the application of a simple in obtaining structure of the immunogenic peptide conjugate.

9 cl, 8 dwg, 7 tbl, 3 ex

 

The technical field to which the invention relates.

The invention relates to medicinal compositions containing the conjugates of albumin-amyloid peptide, and their application, more specifically to their use for the treatment of diseases associated with deposition of amyloid proteins, such as Alzheimer's disease.

The level of technology

Amyloid disease or amyloidosis include a number of illnesses, which have a wide range of external symptoms. When these disorders usually present abnormal extracellular deposition of protein fibrils, known as "amyloid fibrils", "deposition of amyloid or amyloid plaques, which are usually about 10-100 nm in diameter and localized in specific organs or areas of tissue. These plaques consist mainly of natural soluble protein or peptide. Data insoluble deposits consist mainly of lateral aggregates of fibrils, which are approximately 10-15 nm in diameter. Despite the different location, all amyloid deposits have common morphological features, special staining dyes (for example, thioflavin T, Congo red) and have a characteristic property of red-green double refraction in polarized light after staining.

Associated with AMILO the Ohm diseases are characterized by the type of protein, present at the deposition. For example, neurodegenerative diseases such as scrapie, bovine spongiform encephalopathy, a disease of Creutzfeldt-Jakob disease, etc., are characterized by the appearance and accumulation resistant to protease forms of prion protein (referred to as AScr or PGR-27) in the Central nervous system. Similarly, Alzheimer's disease, other neurodegenerative violation, characterized by deposition of amyloid plaques and neurofibrillary glomeruli. In this case, the amyloid plaque and blood vessel formed by the deposition of fibrillar beta-amyloid protein. Other diseases such as diabetes adults (diabetes type II)is characterized by the localized accumulation of amyloid in the pancreas.

Each amyloidogenic protein has the ability to fit in beta folds and form insoluble fibrils that are deposited extracellular or intracellular. Each amyloidogenic protein, despite differences in the amino acid sequence has the same capability of forming fibrils and linking with other elements, such as proteoglycan, amyloid P component of complement. Moreover, each amyloidogenic protein has the amino acid sequence, which, despite their differences, can catalyze the formation of cells with beta-folds. For example, b is the amyloid fibrils is associated with the dead nerve cells and microglia in patients with Alzheimer's disease. When tested in vitro it was shown that beta-amyloid peptide is able to run the activation of microglia (brain macrophages)that would explain the presence of microglia and inflammation of the brain in patients with Alzheimer's disease.

In another type of amyloidosis shown that patients with diabetes type II amyloidogenic protein of ARR induces toxicity in beta-cells of islets in vitro. Therefore, the appearance of fibrils of ARR in the pancreas of patients with type II diabetes could contribute to the loss of beta cells in the islets (of Langerhans) and organ dysfunction.

One of the most famous amyloid diseases is Alzheimer's disease which is a progressive neurodegenerative disease that affects approximately 0.5-1% of the total population of the Western world. Alzheimer's disease is characterized by the deposition of a large number of amyloid plaques in the brain. Assume that the deposition is a cause of the disease, and most approaches to the prevention of Alzheimer's disease is aimed at reducing, removing or preventing the formation of amyloid plaques. The main component of amyloid plaques is the beta-amyloid peptide (β), protein of 40-42 amino acids, which is formed by cleavage of the amyloid protein precursor (UPS).

P the patent application US 20070172496 discloses conjugates, containing the peptide Aβ(33-42), a peptide recognized by a monoclonal antibody and albumin, which are used as immobilized antigens in ELISA assays (ELISA assays) to determine the presence of antibodies to Aβ in the sample obtained from the patient, which is immunized by the complex formed by different peptides selected from a peptide Aβ(1-42), and the so-called ligand-presenting unit LPA.

Thus, in engineering there is a need for immunogenic compositions capable of inducing an effective and prolonged decline in the levels of amyloid in the plasma and to reduce the number of amyloid deposits.

Disclosure of inventions

In the first aspect of the invention relates to a conjugate containing a peptide selected from the C-terminal segment β(1-42), and albumin, intended for use in medicine.

In another aspect the invention relates to compositions containing a conjugate, comprising a peptide selected from the C-terminal segment β(1-42), and albumin, as well as adjuvant intended for use in medicine.

In another aspect the invention relates to a conjugate containing at least immunogenic peptide selected from the C-terminal segment β(1-42), and albumin, or a composition containing at least one immunogenic peptide selected from the C-to the core plot β(1-42), and albumin, as well as adjuvant intended for use to treat or prevent diseases associated with deposition of amyloid proteins.

Brief description of drawings

Figure 1: Reveals the molecular mass distribution (MMD) of conjugates of BSA with immunogenic peptide β(35-42). The average value of the mold corresponds to the molecular mass of the conjugate is 16 immunogenic peptides β(35-42). The largest number of conjugates is placed on the mold in the range of molecular mass conjugates containing from 11 to 21 peptide.

Figure 2 is a diagram illustrating a scheme of immunization injections of peptides β (down arrows) indicating the number of immunization below the line, the collection of blood samples (top arrow) indicating a respective time points of collection of each sample in weeks (W) and the sample collection CFS at week 0 and week 13 (double arrows). (A) is a diagram immunization injections of peptide β(x-42) and (C) - peptide β(x-40). (A) in the left frame of the present scheme, which followed in the treatment of all animals, and in the right frame provide additional interventions carried out only in groups C and D.

Figure 3: (A-C). Rising titers of antibodies to β in the plasma. (A) Titers of antibodies to β in plasma samples is expressed as the equivalent of kolichesvto µg/µl monoclonal antibodies E. C) the Titers of anti-Christ. ate to β in plasma samples Express through their ES. D) the Titers of antibodies to β in plasma samples is expressed by the reciprocal of the maximum dilution of the plasma, in which the absorption is three times higher than the average absorption in the empty holes. Different points in time (in weeks) are on the horizontal axis. Each line represents a single animal, as indicated in the legend. Drugs received by each group of animals, are synthetic peptide "Blue media" (Blue Carrier) and Rapidreel NRA (A), a synthetic peptide - Blue media and Abisco-300 (); synthetic peptide - BSA (bovine serum albumin) and Rapidreel NRA () and synthetic peptide - BSA and Abisco-300 (D). Shorter solid lines correspond areagirls animals (groups a and b), the treatment of which stop at week 13. Animals from groups represented by broken lines and the corresponding individual designation. Animals from group D represented by dashed lines and the corresponding individual designation.

Figure 4 (A-B). The increase of concentration of peptide β in the plasma. (A) represents the growth in the various time points in plasma concentrations of peptide β(1-42). C) represents the growth in the various time points in plasma concentrations of peptide β(1-40). The horizontal axis crosses the Y axis at the point of 3.125 PG/ml Is below 3,125 are as -5 PG/ml for clarity of the graph. Various exact and time (in weeks) are on the horizontal axis. Each line represents a single animal, as indicated in the legend. Each of the four groups (a, b, C and D) receives various vaccine preparations. Shorter solid lines correspond areagirls animals (groups a and b), the treatment of which stop at week 13. Animals from groups represented by broken lines and the corresponding individual designation. Animals from group D represented by dashed lines and the corresponding individual designation.

Figure 5. Growth ratios changes in antibody titers in plasma and concentrations of the peptides relative to preimmune status for each experimental group. Point of time (in weeks) are on the horizontal axis. The dashed line represents the increase in the ratio of plasma concentration of the peptide β(1-42) at various points in time relative to preimmune status (W0 (week 0)). The dotted line represents the increase in the ratio of plasma concentration of the peptide β(1-40) at various points in time relative to preimmune status (W0 (week 0)). The continuous line represents the ratio is higher titers of antibodies to β in plasma at different time points relative to preimmune status (W0 (week 0)). For each correlation value, pending on the vertical axis represent the sum of three animals in the group. The values of the ratio titro the antibody β in the plasma is divided into twenty for clarity of the graphs.

Figure 6. Rising titers of antibodies to β(33-40) in plasma. (A) Titers of antibodies to β in plasma samples is expressed as the equivalent ng/ál antibody β40 (SAR 22). B) Titers of antibodies to β in plasma samples Express through their EU50(concentration giving 50% effect). Different points in time (in weeks) are on the horizontal axis. Each line represents a single animal, as indicated in the legend (A1-A3 are shown in black and B1-B3 - white). Each of the two groups (a and b) receives various vaccine preparations.

Figure 7: is a graph showing the concentration of peptide in soluble form or in the insoluble form in the two groups receiving the vaccination, compared with the control group. Designation Vacc.1 corresponds β-42+BSA+adjuvant Th2-type and designation Vacc.2 corresponds β-42+BSA+adjuvant mixed Th1/Th2-type. Column (1) corresponds to a soluble form β(1-40); (2) soluble form β(1-42); (3) - insoluble form β(1-40) and (4) to the insoluble form β(1-42).

Figure 8 is a graph showing the concentration of peptide in soluble form in vaccinated groups compared with the control group. (A) corresponds to soluble peptides β(1-40) and (C) soluble peptides β(1-42), whereas (1) refers to the sample of the cerebellum; (2) a sample of the frontal; (3) entorhinal and (4) temporal regions of the brain is the mind.

The implementation of the invention

Conjugates corresponding to the invention, are intended for use in medicine

The authors of the present invention have found that the introduction of a conjugate containing a peptide selected from the C-terminal site of beta-amyloid protein (1-42) [Aβ(1-42)], and albumin, unexpectedly causes the formation of antibodies to this peptide and decreased serum levels of proteins Aβ(1-40) and Aβ(1-42), which represent the main components of amyloid plaques in Alzheimer's disease. These results open a new therapeutic approach to treatment, prevention and/or alleviation of diseases associated with deposition of amyloid proteins.

Thus, in one aspect the invention relates to a conjugate containing immunogenic peptide selected from the C-terminal site of Aβ(1-42), and albumin, intended for use in medicine.

Immunogenic peptide. allocated from the C-terminal segmentAβ(1-42)

The term "immunogenic peptide", as used in this context, refers to a peptide which comprises an allele-specific motif, epitope, or other sequence, so that the polypeptide or fragment binds a molecule of MHC (major histocompatibility complex) and induces the response of cytotoxic T-lymphocytes ("CTL") and/or the response of b-cells (e.g., production of antibodies), and/and the and the response of lymphocytes T-helper cells, and/or response type of delayed-type hypersensitivity (DTH) against the antigen from which the selected immunogenic peptide, namely beta-amyloid peptide. Suitable methods for determining whether a given immunogenic peptide shown, in particular, in the examples described in this invention. These methods are based on the ability of these immunogenic peptides to generate antibodies to beta-amyloid in animals after administration of these peptides.

The term "beta-amyloid peptide" is used in this context is interchangeable with the terms of Abete, beta-amyloid protein, "beta", "beta-AR", "Abete peptide" or "β peptide", and he refers to the family of peptides, which represent the main chemical component of senile plaques and vascular amyloid deposits (amyloid angiopathy), detectable in the brain of patients with Alzheimer's disease (AD), down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). In any form of beta-amyloid peptide is a fragment of beta-amyloid protein precursor (APP), which contains a different number of amino acids, usually 39-43 amino acids. "β(1-42)," as used in this context, refers to a peptide of 42 amino acids corresponding to amino acids 672 - 713 RDAs, which obrazets the sequential proteolytic cleavage of amyloid protein precursor using β - and γ-secretase.

Beta-amyloid peptides are usually referred to as Aβ(x-y), where x represents the number of amino acids of aminocore beta-amyloid peptides and represents the number of amino acids of carboxylic. For example, Aβ(1-40) is a beta-amyloid peptide, aminocore which begins with amino acid number 40, the sequence shown in SEQ ID NO:1.

In the context of the present invention, the term "peptide selected from the C-terminal segment β(1-42), is intended to refer to peptides having from 2 to 40 amino acid residues, including part or all of the C-terminal site of Aβ(1-42). The term also encompasses peptides containing areas of significant analogy with the C-terminal site of Aβ(1-42), such as structural variants.

The term "substantial analogy" means that the sequence of the two peptides with optimal alignment have at least 50 percent sequence identity, preferably at least 60 percent sequence identity, more preferably at least 70 percent sequence identity, more preferably at least 80 percent sequence identity, more preferably at least 90 percent sequence identity, more preferably at least 95 PR is virtually all types of sequence identity or more (e.g., 99 percent sequence identity). Preferably, when the position of the residues that are not identical differ by conservative substitutions of amino acids. The term conservative substitutions of amino acids refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is a glycine, alanine, valine, leucine and isoleucine; a group of amino acids having a side chain aliphatic radical is hydroxyl, is a serine and threonine; a group of amino acids having amesterdam side chains, represents asparagine and glutamine; a group of amino acids having aromatic side chains is a phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains, represents lysine, arginine and histidine, and a group of amino acids having sulfur-containing side chains is a cysteine and methionine.

Position of residues that are not identical may also be composed of peptides, including unnatural amino acids or their derivatives. Counterparts, as a rule, differ from natural peptides on one, two or more positions, often as a result of conservative substitutions.

Some analogs also include non-natural amino acids or modifications of the N - or C-terminal AMI is ocelote in one, two or more positions. Examples of unnatural amino acids without limitation are D-amino acids, alpha,alpha-disubstituted amino acids, N-alkylaminocarbonyl, lactic acid, 4-hydroxyproline, gamma-carboxyglutamate, Epsilon-N,N,N-trimethyllysine, Epsilon-N-acetylized, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, omega-N-methylarginine and itaporanga acid.

In a specific embodiment, conjugation, corresponding to the invention, the peptide selected from the C-terminal site of Aβ(1-42) selected from Aβ(35-42) (SEQ ID NO:2), β(33-42) (SEQ ID NO:3) and β(33-40) (SEQ ID NO:4).

Aβ(35-42)", as used in this context, refers to a peptide of 8 amino acids corresponding to the last 8 amino acids of Aβ(1-42).

"Aβ(33-42)", as used in this context, refers to a peptide of 10 amino acids corresponding to the last 10 amino acids of Aβ(1-42).

“β(33-40)", as used in this context, refers to a peptide of 8 amino acids corresponding to amino acids 33 to 40 Aβ(1-42).

Peptides isolated from the C-terminal segment β(1-42), including peptide linker group, it is possible to synthesize standard chemical methods of peptide synthesis in solid or liquid phase. A brief description of solid-phase methods can be found in the monographs by Stewart and Young (1963) Solid Phase Peptide Synthesis (Tverdofazno the th peptide synthesis), W. H. Freeman Co. (San Francisco) and Meienhofer (1973) Hormonal Proteins and Peptides (Hormonal proteins and peptides), Academic Press (New York). In terms of the classical methods of synthesis in solution, see the monograph Schroder and Lupke, The Peptides (Peptides), so 1, Academic Press (New York).

Typically, these methods involve the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Usually either the amino-or carboxyl group of the first amino acid protects a suitable protecting group. Then the protected amino acid is either attached to an inert solid support, or used in solution by adding the next amino acid in the sequence having complementary (amino or carboxyl) group, a protected appropriately, and under conditions suitable for the formation of amide linkages. Then the protective group is removed from this newly added amino acid residue and add the following amino acid (protected accordingly), and so on, Once all the necessary amino acids associated with the proper sequence, any remaining protective groups (and any solid basis) sequentially or simultaneously removed by the end of the peptide. With a simple modification of this General method can be added to the growing chain of more than one amino acid at a time, is for example, by linking (in conditions which do not cause racemization of chiral centers) protected Tripeptide with properly protected dipeptide with the formation of Pentapeptide after removing the protection. In a preferred embodiment, the cysteine residue added to the N-end data immunogenic peptides to facilitate data-binding peptides with the molecules of the carrier when using bifunctional reagents capable of reacting with sulfhydryl groups of cysteine.

Albumin

As noted above, one aspect of the present invention relates to a conjugate containing a peptide selected from the C-terminal site of Aβ(1-42), and albumin, intended for use in medicine.

As used in this context, the term "albumin" refers to the most common protein in blood plasma, having a molecular weight of approximately from 65 to 67 KD in its Monomeric form, depending on the species from which it is selected. The term "albumin" is used interchangeably with the term "serum albumin", ion does not identify the source of albumin, which forms a conjugate with modified peptides corresponding to the invention. Thus the term "albumin"as used in this context may refer either to the albumin, purified from natural IP the student, such as blood or serous fluid, or it may refer to chemically synthesized or recombinante received albumin. In a different implementation variants of albumin or derivative of native albumin can be used to form conjugates corresponding to the invention. In some embodiments, the implementation of albumin is a serum albumin mammals or variant or derivative. Non-limiting examples of albumin mammals, which can be used include human, bovine, sheep, goat, rabbit, cat, dog, pig albumin, albumin primates or rodents. In a preferred embodiment, albumin mammal is a human albumin.

In one embodiment, human albumin purified from blood or serous fluid. For example, the albumin can be cleared from the sera or plasma of patients or laboratory animals, often using bovine albumin, but it can also be extracted from sera obtained from other animals (chicken, pig, rabbit, etc), using any standard method, such as fractionation by cold ethanol method Kona (see Cohn EJ, et al., J Am Chem Soc 1946; 68:459-75) or the method of Kistler and Nitschmann (see Kistler P and Nitschman HS., Vox Sang 1962; 7:414-24) or chromatographic purification (see Bergloff JH. et al. in the monograph edited by Curling JM, Separation of Plasma Proteins (Separation of plasma proteins), Uppsala: Pharmacia, 1983; 51-8) or a combination of fractionation cold ethanol and chromatographic purification. Albumin can also be obtained from egg protein (ovalbumin). Another type of albumin, albumin storage, can be extracted from the seeds of some plants (e.g. soybean).

In another embodiment, the albumin is a recombinant albumin. In a specific embodiment, the albumin is a recombinant human albumin (in this context referred to as "RFA"). In various embodiments, the implementation of the RFA can be obtained in the body of a mammal or memleketim. In one embodiment, the ablation receive in the body of memleketim. Examples of organisms of memleketim that you can use to obtain the RFA include, but not limited to yeast, bacteria, plants, fungi and insects. In one embodiment, the ablation receive in the whole plant or the whole mushroom. In another embodiment, RFA get in cultured plant cells, cultured cells of fungi or cultured insect cells. In another embodiment, the ablation receive in the body of a mammal, otlichno is from a person or in cells of a mammal other than man. Examples of mammals, excellent tcheloveka, which you can use to obtain the RFA include, without limitation belonging to one of the following orders: the order of the Bovidae, order Canidae, order Suidae, order Rodentia, the order Lagomorpha and order Primates (excluding humans). In a particular embodiment, the mammal other than human, which is used for the ablation selected from the group consisting of cow, dog, pig, sheep, goat, rat, mouse, rabbit, chimpanzee and gorilla. In another embodiment, cells of a mammal other than human, used to obtain the RFA, are not limited to bovine, dog, pig, sheep, goat cells, cells of rodents, rabbits or primates other than man. The main advantage of RFA produced

organism other than humans, compared with albumin, purified from human blood or serous fluid, is no product of human nature in the process for RFA. The use of these controlled methods of obtaining gives a more pure product with reduced structural heterogeneity. Previous studies show that there are no significant differences between soluble RFA and human al is the IOR techniques, purified from blood or serous fluid, in terms of biochemical characteristics, the efficiency of injection of radioactive labels and biological behavior in vitro and in vivo. Cm. article Dodsworth et al., 1996, Biotechnol. Appl. Biochem. 24: 171-176. In a specific embodiment, the albumin is a RFA, developed under the trade name RECOMBUMIN(R) (Novozymes Inc., Nottingham, UK). RECOMBUMIN(R) is a recombinant human albumin, which is produced in vitro using the technology of recombinant yeast, in which genetically modified yeast (Saccharomyces cerevisiae) secrete soluble RFA, which is then collected, cleaned and receive in the form of a drug intended for use as a filler for the manufacture of biological products or coatings for medical devices.

Alternative albumin, options or derivatives of albumin, intended for use in the formation of the conjugate corresponding to the present invention, can be obtained from commercial sources, for example, as RECOMBUMIN(R) (Novozymes Inc., Nottingham, UK), PLASBUMIN(R) (Talecris of Vulerabilities, Research Triangle Park, NC), ALBAGEN(R), (New Century Pharmaceuticals, Huntsville, AL), human albumin (Cortex-Biochem, San Leandro, CA), human serum albumin, ZLB Behring (King of Prussia, PA) or ALBREC(R) (Mistubishi Pharma, Japan).

In the context of the present invention, the term "albumin" refers to mu the mu protein, soluble in water, moderately soluble in concentrated salt solutions, and subjected to coagulation when heated (protein denaturation). He has a molecular weight of approximately 65,000, consists of different numbers of amino acids lying in the interval from 609 in most species, including human albumin, to 615 amino acids, like chicken albumin. They all contain 35 cysteine residues that can be used for conjugation immunogenic peptide via disulfide bonds. Proteins albumin, suitable to obtain the conjugates of the present invention include without limitation human albumin (SEQ ID NO:5) or a Mature fragment (amino acids 25-609 SEQ ID NO:5) and bovine albumin (SEQ ID NO:6) or a Mature fragment (amino acids 25-607 SEQ ID NO:6). The albumin used in the present invention also covers structural variants of albumin derived from conservative substitutions of amino acids, as explained above for peptide selected from the C-terminal site of Aβ(1-42).

In some embodiments, the conjugates corresponding to the invention include molecular variants of albumin, such as described in WO 2005/058958, the contents of which are incorporated by reference in its entirety. Variant of recombinant human serum albumin is commercially available in the company New Century Pharma(Huntsville, Alabama) called 1bgn". The albumin used for the formation of the conjugate corresponding to the present invention can be obtained by using methods or materials known to the competent specialists in the field of technology. For example, the albumin can be obtained from commercial sources, for example, Novozymes Inc. (Davis, CA; recombinant human albumin isolated from Saccharomyces cerevisiae), Cortex-Biochem (San Leandro, Calif: serum albumin), Talecris of Vulerabilities (Research Triangle Park, North Carolina; serum albumin), ZLB Behring (King of Prussia. PA) or New Century Pharmaceuticals ille, Ala.: recombinant human albumin isolated from Pichia pastupsilonris).

Variants of albumin may include natural variations resulting albumin polymorphism in the human population. More than 30 obviously different genetic variants of human serum albumin identified by electrophoretic analysis under various conditions. See, for example, article Weitkampetal, Ann. Hum. Genet., 36(4):381-92 (1973); Weitkamp, Isr. J. Med. ScL, 9(9):1238-48 (1973); Fine et al, Biomedicine, 25(8):291-4 (1976); Fine et al, Rev. Fr. Transfus. Immunohematol, 25(2); 149-63, (1982); Rochu et al, Rev. Fr. Transfus. Immunohematol. 31(5):725-33(1988); Araietal, Proc. Natl. Acad. Sc. U. S. A 86(2): 434-8 (1989), the content of which in this context is incorporated by reference in its entirety. In a special embodiment, the invention is a conjugates formed by the molecular variants of albumin.

In some embodiments, the implementation of the conjugates corresponding to the invention include albumin derivatives, which have substantial homology with albumin. For example, the conjugates can be formed with a homologue of albumin having an amino acid sequence at least 75%, at least 80%, at least 85%, more specifically at least 90% and most accurately at least 95% identical to the sequence of albumin. In some embodiments, the homolog of albumin contains a free cysteine.

In some embodiments, the implementation of the conjugates corresponding to the invention contain structural albumin derivatives. Structural albumin derivatives may include proteins or peptides that have activity type activity of albumin, for example, a functional fragment of albumin. In some embodiments, the implementation of a derivative is an antigenic determinant of albumin, i.e., the portion of the polypeptide, which can detect antibody to albumin. In some embodiments, the implementation of the recombinant albumin may be any protein with a large half-period of existence in the plasma, which can be obtained by modification of the gene encoding human serum albumin. As a non-limiting example, the recombinant albumin can in order to keep insertions or deletions in the region of the binding of trace metals albumin, so the binding of trace metals, for example Nickel and/or copper, is reduced or eliminated, as described in U.S. Patent No 6787636, the content of which is incorporated by reference in its entirety. Reduced level of binding of trace metals albumin can be beneficial in reducing the likelihood of allergic reactions to the metal trace elements in patients undergoing treatment composition of albumin.

In some embodiments albumin derivatives include any macromolecule with a large half-period of existence in the plasma, obtained by modification of the protein albumin in vitro. In some embodiments, the implementation of albumin modify fatty acids. In some embodiments, the implementation of albumin modify metal ions. In some embodiments, the implementation of albumin modify small molecules with high affinity to albumin. In some embodiments, the implementation of albumin modify sugars, including, but not limited to, glucose, lactose, mannose and galactose.

Structural albumin derivatives can be generated using any method known to the competent specialists in the field of technology, including, but without limitation, indirect oligonucleotide (site-specifies the s) mutagenesis, alanine scan mutagenesis using the polymerase chain reaction (PCR). Site-specific mutagenesis (see Cotter, Biochem J 237 1-7 (1986), Zoller and Smith, Methods Enzymol 154 329-50 (1987)), cassette mutagenesis, mutagenesis, restriction selection (Wells et al., Gene 34 315-323 (1985)) or other known methods can be performed on the cloned coding albumin DNA with obtaining DNA variant albumin or sequences that encode structural albumin derivatives (see the monograph by Ausubel et al., Current Protocols In Molecular Biology (current protocols in molecular biology), John Wiley and Sons, New York (latest edition), Sambrook et al, Molecular Cloning, A Laboratory Manual, 3d ed (Laboratory manual molecular cloning, 3rd ed.), Cold Spping Harbor Laboratory Press, Cold Spring Harbor, New York (2001), the content of which in this context is incorporated by reference in its entirety.

In some embodiments albumin derivatives include any macromolecule obtained by modifying in vitro protein albumin, with a longer half-life existence in the plasma than the native albumin. In some embodiments, the implementation of albumin modified with one or more fatty acids.In some embodiments, the implementation of albumin modify one or more metal ions. In some embodiments, the implementation of albumin modify one or more small molecules, the region is surrounding high affinity to albumin. In some embodiments, the implementation of albumin modify one or more sugars, including, but not limited to glucose, lactose, mannose and galactose.

Preparations of human serum albumin isolated from serum or obtained by recombinant may contain a heterogeneous mixture of dimercaptotoluene, i.e., "covered" albumin, and mercaptoethylamine, i.e., "uncovered" albumin. The polypeptide of human albumin contains 35 residues of cysteine, 34 of which form 17 stabilizing disulfide bridge. While the cysteine residue in position 34 mercaptamine contains free SH-group of the same residue in dimercaptotoluene contains mixed disulfide, for example, cysteine or glutathione or oxidized metal ions for other adducts, making, thus, the thiol group is less reactive or unavailable. Generally, enrichment mercaptoamines reach by contacting the recombinant albumin with any agent capable of converting oxidized albumin-s34 restored in albumin-Cys, such as dithiothreitol (DTT), thioglycolate acid (TGA) or beta mercaptoethanol (TOGETHER).

In some embodiments of the invention the recombinant albumin may be delicious prior to the conjugation reaction. the read that deliciouse albumin, particularly recombinant albumin produced by yeast can give albumin, which has high portability and stability in relation to the form of conjugates. As a rule, deliciousa albumin can be done using any method and in any conditions, known to the competent specialists in the field of technology that is effective in the recovery plan refermentation glycosylated proteins, such as described in the article Miksiket al (J. Chromatogr. C. Biomed. Sci. Appl., 1997, 699:311-345). Alternative albumin can be diglycinate using enzymatic methods. For example, deliciousa can be done using endoglycosidase N or a mixture of different endoglycosidase.

In another embodiment, the recombinant albumin can be further processed with the aim of preferred specificity of conjugation, i.e. to reduce the probability of formation of conjugates of non-ys. For example, you can make contact recombinant albumin with agents that chemically block the remains of human serum albumin, which, as you know, is the formation of covalent adducts. You can use any agent that is known in the art is able to block the reaction centers in the albumin than Cys3. In some embodiments, the implementation of the agent blocks the lysine residues. Albumin contains different number of lysine residues (e.g., 60 in human albumin and 61 in bovine albumin), of which 25-30 are on the surface of albumin and may be available for conjugation. Accordingly, in some embodiments, the implementation of the agent blocks the lysine residue of albumin, which is known to be a competent specialist in the field of technology, has the potential ability to form covalent adducts, such as Lys71, Lys19, Lys351, Lys525, Lys541 albumin.

The linker section

A peptide selected from the C-terminal part of Abete(1-42), and albumin can be connected directly or, alternatively, can be connected through one or more linking groups (hereinafter referred to as intermediate molecules or spacer elements group).

In a specific embodiment, conjugation, corresponding to the invention, if at least one immunogenic peptide and albumin are connected by linker section, the specified linker section contains no more than one attached immunogenic peptide.

In another specific embodiment, conjugation, corresponding to the invention, the linker section connecting at least one immunogenic peptide and albumin contains sistei is, and more preferably, when the cysteine is at the N end of the immunogenic peptide.

In some embodiments the linking group is a biocompatible polymer, for example a peptide or alkyl, or alkoxysilane polymer. In a specific embodiment, the linking group is a peptide having a labile chemical bond which is cleaved by the enzyme, or fissile-specific chemical conditions, for example under acidic conditions. In one embodiment, the modified peptide contains a reactive group covalently linked to the peptide via one or more linking groups.In some embodiments the linking group includes one or more reactive groups, typically one linking group. In some embodiments the linking group has a length of 1-100 atoms. As described in this context, the length of the linking group expressed through the number of atoms in the shortest chain of atoms between the groups linked by a linking group. In some embodiments the linking group is from 1 to 100 atoms, from 1 to 80 atoms, from 1 to 60 atoms, from 1 to 50 atoms, from 1 to 40 atoms, from 1 to 30 atoms, from 1 to 20 atoms, from 10 to 20 atoms, or from 5 to 15 atoms. In cases when there is more than one linking group, Dan is haunted linking groups may be the same or different linking groups. The linking group can be attached to a peptide selected from the C-terminal part of Abete(1-42), process or technology known to the competent specialists in the field of technology. Examples of methods or technologies described in U.S. Patent No. 6849714, the content of which in this context is incorporated by reference in its entirety.

Linking groups can contain one or more alkyl groups such as methyl, ethyl, propyl, butyl, etc., alkoxygroup, alkenyl groups, etkinlik groups or amino groups, substituted alkyl groups, cycloalkyl groups, polycyclic groups, aryl groups, polyarylene groups, substituted aryl groups, heterocyclic groups and substituted heterocyclic groups.

In some embodiments the linking group may be selected from linking groups, including the amino group and carboxypropyl, including, but not limited to, AEA, AEEA and OA. In some embodiments the linking group may be a polymer AEA, having a length of 1 to 100 atoms. In some embodiments the linking group may be a polymer of AEEA, having a length of 1-100 atoms. In some embodiments the linking group may be a polymer OA, having a length of 1-100 atoms. Illustrative note the factors linking groups include monomers, dimers, trimers, tetramer, pentamers, hexamers, septenary, oktamery, nonamer, decamera, andcamera, dodecameric residues of glycine, lysine, glutamate, isoleucine or arginine, AEA, AEEA or OA, for example, dimer OA (OA-OA-), or trimer OA (OA-OA-OA-), or any combination of them (for example, any combination of Glyn, LySn, OA, AEA or EEA, where n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12). In some embodiments, the implementation of the linking group has a single lysine. The only lysine can be modified to associate it with the reactive group directly or through one or more linking groups. For example, a group may be associated with a reactive group or one or more additional linkers through, for example, the Epsilon-amino group of the side chain. Examples of these linkers, including the only one lysine, have, for example, the sequence (monomer)andTo(monomer)b(Monomer)cand (monomer)dTo where each of a, b, C and d means integer greater than or equal to one, for example one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more. In some examples a and b can be identical, whereas in other examples a and b are unequal. For example, can mean three and b can mean four to obtain the linking group has the following consequences is the sequence: (OA) n(OA)n, (OA)n(AEA)n(G)nK(OA)n, (OA)nK(G)nwhere n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In the case when the reactive group is located in the linker between the two TSR, total length between the two TSR, as a rule, will not exceed 100 atoms. Group attached to the linker group, is not considered part of the linker between the two TSR.

The linking group may include any combination of the above biocompatible polymers. For example, polyglycine linker can be combined with one or more monomers AEA, AEEA or OA in any configuration. In one embodiment, polyglycidyl the linker is attached to one or more monomers AEA, AEEA or OA or the N-or C-terminal site of the first or the last glycine residue polyglycine linker. Alternative one or more monomers AEA, AEEA or OA injected between glycine residues in polyglycidol the linker. In specific embodiments, the implementation of the reactive group (for example, MPA, GMBA, NHS, sulfo-NHS, MBS or GMBS) attached to the peptide via one or more linking groups, including, for example, polyglycine linker, polypyrimidine linker, AEEA, AEA or OA, or any combination. In some embodiments, when the reactive group attached to the peptide via more than one linking group, each the linking group may be independently selected from the group consisting, as a rule, from polyglycine, polylysine, AEA, AEEA and OA. In embodiments, the implementation of a number of linking groups (e.g., Monomeric units of the polymer) is from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12. When there is more than one linking group, the linking group may be the same or different linking groups. For example, you can use any combination of one group of polyglycine, polylysine, AEA, AEEA and/or OA in any order. In one embodiment, reactive group, typically MPA, is attached to the peptide via 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 polyglycine, polylysine, AEA, AEEA or OA linking groups, which are organized in tandem. In another embodiment, the reactive group, typically MPA, is attached to the peptide via 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 polyglycine, polylysine, AEA, AEEA or OA linking groups, which are arranged in a branched configuration.

In another embodiment, the linking group is a peptide group, which can act as a hinge section between peptide selected from the C-terminal segment β(1-42), and albumin, enabling them to move more independently from each other, while they maintain their own individual remerol form. In this sense, the preferred unnatural intermediate amino acid sequence corresponding to the invention, it will be the hinged area, characterized by structural plasticity, providing the possibility of this movement, or unnatural flexible linker. The flexible linker may be a flexible linker peptide with a length of 20 amino acids or less. In a more preferred embodiment, the linker peptide contains 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. In a preferred embodiment of the invention the specified flexible linker is polyglycine linker, but not limited to, polyglycidol, polyglutamines, politicaleconomy, poliropilennovoy or other suitable linking group comprising two or more amino acids. In some examples, the amino acid linking group may include at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid residues, such as residues of glycine or lysine. Polyglycidyl the linker may include one or more different residues (e.g., lysine residues), entered in any configuration, for example, near the end of the N - or C-terminal segment or in the middle of the chain residue of glycine. In other vari is ntah implementation polyglycine linker is combined with one or more monomers AEA, EEA or OA in any configuration. In one embodiment, polyglycidyl the linker is attached to one or more monomers AEA, AEEA or OA or the N-or C-end of the first or last residue of glycine in polyglycidol the linker. Alternative one or more monomers AEA, AEEA or OA injected between glycine residues in polyglycidol the linker. In the examples, when polyglycine used as a linker, polyglycine may include a single lysine to obtain the free Epsilon-amino group capable of reaction with another linker or protein. Examples of this polyglycine, which includes amino acid, for example, one lysine, have, for example, the amino acid sequence of (G)aK(G)bTo(G)cand (O)dTo where each of a, b, C and d represents an integer number greater than or equal to one, for example one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more. In some examples a and b can be identical, whereas in other examples a and b are unequal. For example, can mean three, b can mean four. In some examples, a single lysine in polyglycidol the linker can be linked to additional groups, for example, a linking group, the reactive group or the residue of the reaction of the group. For example, the lysine residue may be attached to gnome or more additional linkers through, for example, the Epsilon-amino group of the side chain.

Possible examples of the linker/spacer elements sequences include SGGTSGSTSGTGST (SEQ ID NO:7), AGSSTGSSTGPGSTT (SEQ ID NO:8) or GGSGGAP (SEQ ID NO:9) and GGGKGGGG (SEQ ID NO:10). Sequence data used for binding designed superspray with other protein domains (see Muller, K. M., Arndt, K. M. and Alber, T., Meth. Enzymology, 2000, 328: 261-281). Preferably, when the specified linker contains or consists of a sequence of amino acids GGGVEGGG (SEQ ID NO: 11).

The effect of the linker section is to provide a space between the peptide selected from the C - terminal site of Aβ(1-42), and albumin. It is, therefore, ensures that the presence of albumin does not affect the secondary structure of a peptide selected from the C-terminal site of Aβ(1-42), and Vice versa. Preferably, when the spacer is a peptide nature. Preferably, when the linker peptide contains at least two amino acids, at least three amino acids, at least five amino acids, at least ten amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids or priblizitel is but 100 amino acids.

In addition, the linker can be associated with components, flanking peptide selected from the C-terminal site of Aβ(1-42), and albumin by covalent bonds, and preferably, when the spacer is basically non-immunogenic and/or does not contain a single cysteine residue. Likewise preferably, when the three-dimensional structure of the spacer is a linear or substantially linear.

The linker can include residues 53-56 tetractine, forming a β-fold tetractine and residues 57-59, formative stage in tetractine (see Nielsen, C. C. et al., FEBS Lett. 412: 388-396, 1997).

The sequence of segment represents a GTKVHMK (SEQ ID NO:12).

This linker has the advantage that, when present in native tetractine, he binds the domain of the trimerization domain CRD and, consequently, suitable for binding of the trimerization domain with another domain in General. Moreover, do not expect that the resulting design will be more immunogenic than the design without the linker.

Alternative suppositionally from connecting circuit 3 human fibronectin can be selected as a linker, corresponding to amino acids 1992-2102 (numbering SWISSPROT, entrance R). Preferably, when used suppositionally PGTSGQQPSVGQQ (SEQ ID NO:13)corresponding to amino acids No 2037-2049, inside and Yes the Noah of suppositionally preferred fragment GTSGQ (SEQ ID NO:14), corresponding to amino acids 2038-2042. The advantage of this construction is that it is not very susceptible to proteolytic cleavage and not very immunogen, since fibronectin is present in plasma in high concentrations.

An alternative basis of an appropriate peptide linker may be a sequence of 10 amino acid residues of the upper hinged section of mouse lgG3. This peptide (PKPSTPPGSS, SEQ ID NO: 15) is used to produce antibodies, demonizovana by supercoiling (see article Pack P. and Pluckthun, A., 1992, Biochemistry 31:1579-1584), and it can be used as spacer elements peptide corresponding to the present invention. The corresponding sequence of the upper hinge area of the human lgG3 may be even more preferable. Do not expect that the sequence of the human lgG3 will be immunogenic to humans. In a preferred embodiment, the linker peptide is selected from the group of the peptide sequence ARAECERUS (SEQ ID NO: 16) peptide sequence GAP.

In a specific embodiment, the linker section contains the following groups: -N(CH2-)2, -N< or -- NHCH(CH2-)2.

In a specific embodiment, the conjugate, corresponding to the invention, has the following structure:

X-L -R<CO-LB-NH-P-CO-YCO-LB-NH-P-CO-Y

where

R represents-N(CH2-)2, -NHCH< or -- NHCH(CH2-)2,

X represents a hydrogen or a peptide group, and

LAoptionally present and represents an amino acid or a peptide containing at least 2 amino acid residue,

LBoptionally present and represents an amino acid or a peptide containing at least 2 amino acid residue,

P is a peptide selected from the amyloid proteins or proteins substantially close amyloid protein of full length or fragments

Y is HE or NH2,

and their pharmaceutically acceptable salts.

Conjugation of a peptide selected from the C-terminal segmentAβ(1-42), and albumin

With the availability of a sufficient amount of a peptide selected from the C-terminal site of Aβ(1-42), and albumin conjugate, corresponding to the invention, is formed by contact of both components under conditions suitable for formation to Alentova complex between them.

Regardless of whether immunogenic peptide and albumin directly or through a linking group, the invention provides the possibility of attaching a peptide through its M-the end (end view), and attaching the peptide to a molecule of albumin through its C-end (N-terminal representation). In a preferred embodiment, the immunogenic peptide is presented on the C-end of the molecule to albumin. The term "C-terminal view", as used in this context, refers to peptides that are attached to a protein-carrier (albumin) through their N-terminal section, so that the end remains available for recognition by the immune system.

When the peptide and a molecule of albumin linked through a linker is typically achieved through the use of homobifunctional reagents that can react either with a free α-amino group at the N-end of the peptide and with the primary amino groups in the molecule of albumin or lysine groups, or with a free α-amino group at the N-end. Suitable homobifunctional reagents for binding of primary amino groups immunogenic peptide and albumin include without limitation dialdehyde, such as glutaraldehyde, glyoxal, succinaldehyde, ethylsuccinate, 2-methylglutaronitrile, 3-methylglutaronitrile, ADI is aldehyde, etc.

Albumin is in contact with a peptide selected from the C-terminal site of Aβ(1-42), in a solution containing a final molar ratio of peptide and albumin approximately 0.1:1 to about 10000:1. In some embodiments, the final molar ratio of approximately 7500:1, 5000:1, about 2500:1, about 1000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 75:1, about 50:1, about 25:1, about 10:1, about 7.5:1, about 5:1, about 2.5:1 or approximately 1:1. In some embodiments, the final molar ratio of about 0.1:1 -1:1. In some embodiments, the final molar ratio is approximately 0,1:1, 0,2:1, 0,3:1, 0,4:1, 0,5:1, 0,6:1, 0,7:1, 0,8:1, 0,9:1.

Linking the first and second components of the conjugate takes place through the reaction of the group that is attached to one peptide selected from the C-terminal domain of Aβ(1-42). Reactive group chosen according to their ability to form stable covalent bond with albumin, for example, by reaction with one or more amino groups, hydroxyl groups or thiol groups on the albumin. Preferably, when the reactive group reacts with only one amino group, hydroxyl GRU who sing or thiol group on the albumin. Accordingly, the reactive group can be associated with any Central peptide or linking group which, in the opinion of a competent specialist in the field of technology is appropriate. In some embodiments the reactive group is linked to the skeleton of the peptide or derivative. In some embodiments the reactive group attached to the N-end, for example, N-terminal amine of the peptide or derivative. In some embodiments the reactive group attached to the C-end, for example, C-terminal carboxyl peptide or derivative. In some embodiments the reactive group is attached to the side chain of the peptide or derivative, such as hydroxyl, thiol, amino or carboxyl side chain of the peptide or derivative. In specific embodiments, the implementation of the reactive group attached to the Epsilon-amino group of the lysine side chain of the peptide or derivative. In specific embodiments, the implementation of the reactive group attached to a cysteine residue located at the N-end or the end of the peptide.

For the formation of covalent bonds with functional groups on the protein as a chemical reactive group can be used with a wide range of active carboxyl groups, particularly esters. Carboxyl group, as a rule, turn into the reaction intermediate the products, such as N-hydroxysuccinimide (NHS) or maleimide that are sensitive to the effects of amines, thiols and hydroxyl functionalities on the protein. The introduction of the NHS and maleimide reactionary groups in the peptide can be accomplished by using a bifunctional linking agents, such as maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyrylcholine ester (GMBS), dithiobis-N-succinimidylester (DTSP), N-Succinimidyl 3-(2-pyridylthio) (SPDP), Succinimidyl-TRANS-4-(maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), succinimidylester (SATA), benzophenone 4-maleimide, lambda'-((2-pyridyldithio)ethyl)-4 - azidoaniline (PEAS; AET). Data bifunctional linkers will activate either the carboxy or amino group on the peptide-based selection of protective groups.

Alternate introduction of maleimide in the peptide can be performed when using binding agents such as N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-{3-dimethylaminopropyl) carbodiimide, hydrochloride (EDAC), etc., for activation of derivatives, such as maleimidomethyl acid, [2-[2-[2-maleimidopropionamide

(ethoxy)ethoxy]acetic acid and the subsequent reaction with the amine in the peptide. Similar agents such as DCC and EDAC, could also be used for introduction of derivatives, such maleimidomethyl, carboxypropyl on the peptide.

Per the ranks amines are the main targets of the NHS esters. Available Epsilon-amino group located at the N-end proteins react with NHS esters. However, the Epsilon-amino group on the protein can be undesirable or unavailable for binding to the NHS. Although five amino acids contain nitrogen in the side chains, only the Epsilon-amine of lysine reacts substantially with NHS esters. Amide bond can be formed when the conjugation reaction of the NHS ester takes place with primary amines, releasing N-hydroxysuccinimide. Data succinimidylester reactionary groups in this context refers both to Succinimidyl group.

In specific embodiments, the functional group on the albumin is a single free thiol group located at amino acid residue 34 (Cys34), and chemically reactive group is maleimidomethyl group, such as MPA. MRA means maleimidomethyl acid or maleimidomethyl.

Data maleimidomethyl group mean in this context as maleimide.

In some embodiments, the implementation of conjugates formed by the methods described in this context, contain albumin covalently associated with Succinimidyl or maleimido on therapeutic peptide. In some embodiments, the implementation of the amino, hydroxyl or thiol group of the albumin Kowal is NTO associated with Succinimidyl or maleimido on therapeutic peptide. In some embodiments, the implementation of the thiol of cysteine 34 of albumin covalently linked to [2-[2-[2-maleimidopropionamide(ethoxy)ethoxy]acetamide linker on the Epsilon-aminosidine therapeutic peptide.

In a specific embodiment, the reactive group is a one reactive group of the MPA, is attached to the peptide, optionally through a linking group, for one particular amino acid, and MPA covalently attached to albumin one amino acid residue of albumin, preferably the cysteine 34. In a preferred embodiment, the albumin is a recombinant human albumin.

In some embodiments reactive group can be attached to any residue of a therapeutic peptide, suitable for joining this reactionary group. The rest can be a terminal or an internal residue of the peptide. In some embodiments reactive group can be attached to carboxylic or aminobenzo peptide. In suitable embodiments, the implementation of the reactive group attached to the same center of the peptide. This can be achieved with the use of protective groups known to the competent specialist in the field of technology. In some embodiments derived therapeutic peptide may contain OST the current entered for attaching a reactive group. Useful in merger remnants include, but are not limited to, residues of cysteine, lysine, aspartate and glutamate. The rest you can enter inside or at the end of the peptide, for example, on the balance of N-terminal amino acids through free alpha aminocore. In some embodiments the reactive group attached to an internal lysine residue. In some embodiments the reactive group attached to the terminal lysine residue. In some embodiments the reactive group attached to aminoclonazepam the lysine residue. In some embodiments the reactive group attached to carboxykinase the lysine residue, such as lysine residue on carboxylic therapeutic peptide.

In other embodiments, the implementation of an activated disulfide group can be attached to the cysteine or analog of cysteine therapeutic peptide by way of the preferential formation of intermolecular disulfide bonds schema-based selective activation of the thiol. The described methods based on selective activation of one thiol activating group and subsequent reaction with the second free thiol with the formation of asymmetric disulfide bonds selectively between proteins or peptides to facilitate the resolution of p is oblama low output due to the formation of the symmetrical disulfide bonds. Cm. monograph D. Lambdandreu et al "Methods in Molecular Biology" (Methods in molecular biology) (Ed. by M. W. Pennington and C. M. Dunn, T. 35, page 91., Humana Press, Totowa. N. J., (1994).

Preferably, when the data activating groups are groups on the basis of pyridylsulfonyl (see M. S. Bernatowicz el id., Int.J. Pept. Protein Res. 28: 107(1986)). Preferably, when using 2,2'-dithiodipyridine (DTDP) (see Carlsson et al., Diupsilonchem. J. 173:723(1978); L. H. Kondejewski et al., Bioconjugate Chem., 5:602 (1994) or 2,2'-dithiobis(5-nitropyridine) (NPYS) (see J. Org. Chem., 56:6477 (1991)) or N-Succinimidyl 3-(2-pyridyldithio) (SPDP). In addition, 5,5'-dithiobis(2-nitrobenzoic acid) (reagent Llama) or 6,6'- deiodination acid can be used as the activating groups.

In accordance with these methods group, activating disulfide bonds to first react with therapeutic peptide containing cysteine or similar cysteine, in conditions of excess activating group. These conditions are highly conducive to the formation of medicinal compounds containing a therapeutic peptide that is associated with an activated disulfide group, mostly without education are linked by a disulfide of homodimeric peptide. After binding assays the resulting peptide is then purified, for example using HPLC (high-performance chromatography with reversed phase. Reaction with a second free thiol of origin Taiwan is the CIO, when the peptide compound reacts with albumin with the formation of a conjugate between drug connection, and serum albumin.

Cysteine or similar cysteine therapeutic peptide in turn contains the S-sulfonate by reaction scheme of sulfatase. In this scheme, therapeutic peptide is first get either synthetic or recombinant means. Then use the reaction sulfatase to connect the S-sulfonate to therapeutic peptide through thiol its cysteine or analog of cysteine and subsequent reactions of sulfatase, the connection of therapeutic peptide is purified, for example, using a gradient column chromatography. Then connection the S-sulfonate is used for forming a conjugate between the connection of therapeutic peptide and blood component, preferably serum albumin.

Modification of therapeutic peptides reactive group for conjugation to albumin will widely vary depending on the nature of the various elements containing therapeutic peptide. Methods of synthesis will be selected so that they are simple, gave high yields and made it possible to obtain a highly purified product. Usually chemically reactive group create at the last stage of peptide synthesis, for example, by esterification of carboxy who enoy group with formation of an active complex of ether. Special methods of obtaining modified insulinotropic peptides described in U.S. Patent No. 6329336, 6849714 or 6887849.

Conjugation of the first and second components of the conjugate, corresponding to the invention can be implemented in various ways. One possibility is the direct conjugation of functional groups with therapeutically active component in a position that does not impair the activity of this component. As understood in the present invention, the term functional group refers to a group of specific atoms in a molecule that are responsible for the characteristic chemical reactions of the specified molecule. Examples of functional groups include, but are not limited to the group hydroxy, aldehyde, alkyl, alkenyl, quinil, amide, carboxamide, primary, secondary, tertiary and Quaternary amines, aminoxy, azide, azo (diimide), benzyl, carbonate, ester, simple ether, Glyoxylic, haloalkyl, haloforms, Imin, imide, ketone, maleimide, isocyanide, isocyanate, carbonyl, nitrate, nitrite, nitro, nitroso, peroxide, phenyl, phosphine, phosphate, phosphono, pyridyl, sulfide, sulfonyl, sulfinil, thioether, thiol and oxidized 3,4-dihydroxyphenylalanine (DOPA).

Another possibility is the conjugation of the first and second components through the use of Homo - or heterobifunctional groups. Beef is nacionalnog group can be konjugierte first peptide, allocated from the C-terminal site of Aβ(1-42), and then konjugierte with albumin, or alternatively it is possible to konjugierte bifunctional group with albumin and then konjugierte her with a peptide selected from the C-terminal site of Aβ(1-42). Illustrative examples of these types of conjugates include conjugates, known as ketone-oxime (described in US20050255042), where the first component of the conjugate contains aminochrome, which is associated with a ketone group present in heterobifunctional group, which, in turn, is linked to the amino group in the second component of the conjugate.

In another embodiment, the agent used for the conjugation of the first and second components of the conjugate, corresponding to the invention, can be subjected to proteolytic, chemical, thermal or enzymatic treatment. Particularly interesting is the use of binding agents, which can be subjected to hydrolysis by enzymes located in the target cell, so that therapeutically active compound is released only inside cells. Examples of the types of binding agents that can be processionary inside the cell, described in WO 04054622, WO 06107617, WO 07046893 and WO 07112193.

The components of the conjugate, corresponding to the invention can be chemically modified, provided that the secondary structure and functionality of ebaycompete remain unchanged. The methods of chemical modification of the polypeptide chain is widely known to the competent specialist in the field of technology and include methods based on the conjugation through the thiol groups present in the molecules of cysteine, the methods based on the conjugation through the primary amino groups present in the molecules of lysine (US6809186), methods based on conjugation via N - and C-terminal groups. Reagents suitable for the modification of polypeptides with in order to be able to link them with other compounds include glutaraldehyde (it allows you to associate the connection with the N-terminal part of the polypeptide), carbodiimide (it allows you to associate the connection with the C-terminal part of the polypeptide), operations esters (for example, MBS, SMCC, which you can activate the N-terminal part of the cysteine groups, benzidine(VAV), which allows you to activate the tyrosine group, periodic, which allows you to activate the carbohydrate group, and proteins that are glycosylated.

In a specific embodiment, the peptide selected from the C-terminal site of Aβ(1-42), contains, in addition, an additional N-terminal Cys.

In another embodiment, the modified peptide

containing whey protein, get in vitro (ex vivo) by covalent joining of the modified peptide to orotocol protein in vitro, so the remainder of the reactive group of the peptide forms a covalent bond with whey protein. In one embodiment, the whey protein is autologous to the patient. In a specific embodiment, whey protein allocate the patient. In some embodiments the selected whey protein the patient's purified from other proteins present in blood and/or blood cells before covalent attach to the modified peptide. In accordance with this embodiment the resulting conjugate is administered to the patient from whom the selected whey protein, or autologous to the patient. In another embodiment, the whey protein is a recombinant protein of whey. Typically, whey protein is a recombinant albumin, the most frequently whey protein is a recombinant human albumin. In a preferred embodiment, the conjugate, corresponding to the invention is formed by contacting the modified peptide containing maleimido, with diastereomer whey protein, usually albumin, in applications where the pH from 6.5 to 7.4, usually with the formation of this stable thioester connection that cannot be broken down in the physiological condition is Yah. In some preferred embodiments, the implementation of whey protein is a recombinant human albumin or recombinant bovine albumin).

In one embodiment, the modified peptide amitirova on the C-terminal part. In another embodiment, the modified peptide is not amitirova on the C-terminal part. The modified peptide conjugate or a compound corresponding to the invention can also include this aminirovanie peptide. In one embodiment, the modified peptide allerban N-terminal part. In another embodiment, the modified peptide reallylove N-terminal part. The modified peptide conjugate, compound corresponding to the invention can also include this acylated peptide.

The binding of immunogenic peptides to albumin via a cysteine residue at the N-end of the peptide allows you to bind with one molecule of albumin as many molecules of the peptide as cysteine residues located in the molecule of albumin. For example, bovine serum albumin (BSA) contains 35 cysteine residues, which may be occupied by so many as 35 peptides modified by N-terminal Cys to obtain a conjugate containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29,30,31, 32, 33, Il 35 molecules of the peptide.

Alternative binding of immunogenic peptides to albumin carried out using homobifunctional reagent capable of reacting with primary amino groups, and the conjugate can include as many molecules of the peptide as lysine residues located in the molecule of albumin. For example, bovine serum albumin (BSA) contains 58 lysine residues, which can be occupied by so many as 58 immunogenic peptides to obtain a conjugate containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58 of the peptide molecules.

Thus, bovine serum albumin, contains 35 cysteine residues and 58 lysine residues, can be attached via named the remains of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 of the peptide molecules. Figure 1 : example of peptide β(35-42) presents the molecular-mass distribution of conjugates of bovine serum albumin with the above number of peptides.

Compositions corresponding to the invention, intended for use in the medical field

Conjugates, CE is relevant to the invention, able to induce immune response of the patient, leading to an increase in the number of antibodies that are specific against β(1-42), and reduction of amyloid load in the serum. However, as is known to the competent specialist, the immune response can be enhanced with the use of adjuvants to improve the antigenicity of the conjugate. Thus in another aspect of the present invention a composition comprising a conjugate, corresponding to the invention, and adjuvant designed for use in medicine.

The term "adjuvant"as used in this context, refers to the immunomodulatory substances, suitable for combining with the conjugate corresponding to the invention, in order to enhance, enhancement, or modulation of the otherwise immune response of the patient without harmful effects on the patient.

Adjuvants exert their immunomodulatory properties through several mechanisms, such as the recruitment of lymphoid cells and induction of cytokines. Adjuvants-cytokines include, but are not limited to granulocyte-macrophage colony-stimulating factor, interleukin-12, GM-CSF, a synthetic analogue of murmillos of the dipeptide or monophosphorylated. Additional examples of adjuvants selected from the group consisting of full beta-blockers, incomplete adjuvant's adjuvant, QS21, a gel of aluminum hydroxide is I, MF59, calcium phosphate, liposyn, saponin, squalene, L121, emulsion of monophosphorylated (MPL), Polysorbate 80, cholera toxin (CT), LTK and LTK63. Preferably, when adjuvants such that are approved for use in humans, such as gel, aluminum hydroxide, calcium phosphate and MF59.

In a more specific embodiment, the adjuvant refers to the type that stimulates type TP2 immune response, such as, for example, a gel of aluminum hydroxide and ART. After induction of response type TP2 dominated by the production of inflammatory cytokines, such as IL-4, IL-10 and beta-TGF (transforming growth factor beta), as well as the production of antibodies of classes lgG1 and lgG2b. Preferred adjuvants for use with the purpose of obtaining a preferential response type TN include, for example, photopolymer (see Guy et al. 1998, Vaccine 16:850-856) and alum (for example, aluminum hydroxide, aluminum phosphate).

In the invention it is possible to use any of the "hydroxide" or "phosphate" adjuvants which are usually used as adjuvants. Adjuvants, known as the "aluminum hydroxide"as a rule, are salts oxyhydroxide aluminum, which are usually at least partially crystalline. Adjuvants, known as "phosphate of aluminium, as a rule, are hydroxyphosphate aluminum, often containing small amounts of the sulfate (i.e., hydroxyphosphate aluminum sulfate). You can get them by deposition, and reaction conditions, and the concentration in the deposition process affect the degree of substitution of hydroxyl phosphate in salt.

Fibrous morphology (for example, as seen in the photos, obtained by using transmission electron microscope) typical adjuvants on the basis of aluminium hydroxide. P1 adjuvants on the basis of aluminium hydroxide is usually about 11, i.e., himself adjuvant has a positive surface charge at physiological pH. For adjuvants based on aluminum hydroxide described adsorption capacity of 1.8-2.6 mg protein/mg Al3+at pH 7.4.

Adjuvants based on a phosphate of aluminum, typically have a molar ratio of PO4/Al of 0.3 to 1.2, preferably between 0.8 and 1.2, and more preferably from 0.95±0,1. The aluminum phosphate is usually amorphous, especially in hydroxyphosphate. A typical adjuvant is an amorphous hydroxyphosphate aluminium molar ratio RHO4/Al from 0.84 and 0.92, including 0.6 mg Al3+/ml. of aluminum Phosphate will generally be in the form of particles (for example, to have a plate-like morphology, as seen in the photographs obtained using a transmission electron microscope. Typical diameters of the particles are in the range of 0.5-20 μm (for example, priblizitel is but 5-10 μm) after adsorption of any antigen. For adjuvants on the basis of aluminum phosphate described adsorption capacity of 0.7-1.5 mg protein/mg Al3+at pH 7.4.

The point of zero charge (PZC) of aluminum phosphate is inversely proportional to the degree of substitution of phosphate by hydroxyl, and the degree of substitution can vary depending on the reaction conditions and concentrations of reagents used to obtain salt by precipitation.

PZC is also changed when changing the concentration of free phosphate ions in solution (more phosphate=more acidic PZC) or by adding a buffer such as his-tag buffer (makes PZC more basic). Phosphates of aluminum, used in accordance with the invention will generally be PZC from 4.0 to 7.0, more preferably from 5.0 to 6.5, for example approximately 5,7. Suspension aluminum salts used to obtain the compositions corresponding to the invention may contain a buffer (e.g. phosphate or his-tag or Tris-buffer), but this is not always necessary. Preferably, when the suspension is sterile and apyrogenic. Suspension may include available water phosphate ions, for example, present in a concentration of from 1.0 to 20 mm, preferably from 5 to 15 mm and more preferably approximately 10 mm. Suspensions may also include sodium chloride.

In the invention it is possible to use a mixture of a hydroxide of aluminum is of, and aluminum phosphate. In this case, the aluminum phosphate may be more than hydroxide, for example, when the mass ratio of at least 2:1, for example >5:1, >6:1, >7:1, >8:1, >9:1, etc.,

Preferably, when the concentration of Al4+in compositions intended for administration to the patient is less than 10 mg/ml, for example, <5 mg/ml <4 mg/ml <3 mg/ml <2 mg/ml <1 mg/ml, etc., the Preferred range is from 0.3 to 1 mg/ml to a Maximum of 0.85 m g/dose is preferred.

On the other hand, adjuvant refers to the type that stimulates a Th1 type immune response. As used in this context, the term adjuvant type 1 or Th1 adjuvant designed to determine the adjuvant, which stimulates a Th1 response (type 1) (or the answer that polarized or smashin in the direction of the response type 1 with a relatively weak response Th2 (type 2)). May require Th1 immune responses (characterized by the production of gamma-interferon (γ-IFN) and are associated with protective immunity to viruses and intracellular bacteria), and, consequently, in another specific embodiment, the adjuvant refers to the type that stimulates the immune response of the Th1 type. Preferred adjuvants that are used to generate predominantly Th1 type response may be selected from the group consisting of complete adjuvant the Blockers who, monophosphorylated And 3-des-O-acylated monophosphorylated A (3D-MPL), aluminium salts, CpG oligonucleotides, immunostimulatory DNA sequences, saponin, Montanide ISA 720 (Seppic, France), SAF, ISCOMS (CSL), MF-59 (Chiron), the SBAS-3, SB. Other adjuvants are preferred for Th1 include SAF (Chiron, Calif., United States), series adjuvants SBAS (e.g., SBAS-2 or SBAS-4, manufactured by SmithKline Beecham, Rixensart, Belgium), detox (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylphosphonic 4-phosphates (AGPs), such as described in Patents CLLlANos. 6113918 and 6355257 described in this context by reference in its entirety.

The invention also provides the use of combinations of adjuvants that stimulate both types, Th1 and Th2. In a preferred embodiment, the adjuvant, which stimulates both types, Th1 and Th2, is a saponin. Saponins are a heterogeneous group of Sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the tree Quillaia saponaria Molina widely investigated as adjuvants. Saponin can also be commercially obtained from Smilaxornata (sarsaparilla), Gypsophilla paniculata (tumbleweeds or gypsophila paniculate), and Saponaria officinalis (soapwort). Adjuvant drugs include saponin purified p is aparati, such as QS21, and lipid drugs, such as ISCOMs. Composition of saponin purified using HPLC and RP-HPLC (HPLC with reversed phase). Using these methods identify specific purified fractions, including QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C. Preferably, when the saponin QS21 is a. The saponin preparations may also contain a Sterol, such as cholesterol. Combinations of saponins and cholesterol can be used for the formation of unique particles called immunostimulating complexes (ISCOMs). ISCOMs typically also include a phospholipid, such capovaticano or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, when ISCOM includes one or more components of the QuilA, SHE QHC. ISCOMs are described in detail in references 45-47. Optional ISCOMs can be free from additional detergent. A review of the development of adjuvants on the basis of saponins can be found in articles Barr et al. (Advanced Drug Delivery Reviews, 1998, 32:247-271) and Sjolanderet et al. (Advanced Drug Delivery Reviews, 1998, 32:321-338).

Conjugates corresponding to the invention may have improved pharmacokinetic properties compared with the unconjugated peptides. Pharmacokinetic properties of the peptide include, for example, its rate of absorption and excretion, his profile distribution in tissues, its rate of metabolism and E. what about the toxicity. Typically, the conjugates corresponding to the invention have a reduced rate of excretion and/or increased half-life existence in the bloodstream in vivo compared with conjugialis peptides.

Preferred conjugates intended for use in the present invention include:

PeptideMediaAdjuvant
NH2-MVGGWIA-COOH (Aβ35-42)AlbuminRapidreel NRA
NH2-GLMVGGWIA-COOH (β33-42)AlbuminRapidreel NRA
NH2-MVGGWIA-COOH (β35-42)AlbuminAbisco
NH2-GLMVGGWIA-COOH (β33-42)AlbuminAbisco
NH2-CMVGGWIA-COOH (AS35-42Cys)AlbuminRapidreel NRA
NH2-CGLMVGGWIA-COOH (Aβ33-42Cys)AlbuminRapidreel NRA
NH2-CMVGGWIA-COOH (Aβ35-42Cys)Abisco
NH2-CGLMVGGWIA-COOH (Aβ33-42Cys)AlbuminAbisco
NH2-CGLMVOGW-COOH (Aβ33-40Cys)AlbuminRapidreel NRA
NH2-CGLMVGGW-COOH (Aβ33-40Cys)AlbuminAbisco

Methods of treatment, corresponding to the invention of

As already mentioned in the beginning of the description, composition, corresponding to this invention, as shown, is effective in the treatment of a disease associated with deposition of amyloid proteins. Consequently, in another aspect, the invention relates to a conjugate containing a peptide selected from the C-terminal site of Aβ(1-42), and albumin, or a composition comprising a peptide selected from the C - terminal site of Aβ(1-42), and albumin, as well as adjuvant intended for the treatment of diseases associated with deposition of amyloid proteins.

In another aspect the invention relates to a conjugate containing a peptide selected from the C-terminal segment β(1-42), and albumin, or a composition comprising a peptide selected from the C-terminal segment β(1-42), and albumin, as well as adjuvant, designed for the production of Lekarstvo the th means for treatment of the disease, associated with deposition of amyloid proteins.

In another aspect, the invention relates to a method of treatment of a disease associated with deposition of amyloid proteins, providing an introduction to the needy in the subject conjugate containing a peptide selected from the C-terminal site of Aβ(1-42), and albumin, or a composition comprising a peptide selected from the C-terminal site of Aβ(1-42), and albumin, as well as adjuvant.

As used in this context, the terms "treat", "treating" and "treatment" refers to alleviating one or more symptoms associated with a disease that is the result of introducing a therapeutically effective amount of a composition corresponding to the invention, or containing a pharmaceutical preparation to a patient in need of specific treatment. Thus, the term "treatment"as used in this context, covers the treatment of diseases, disorders or condition of a mammal, in particular human, and includes: (a) preventing the disease or condition in a subject which may be predisposed to the disease or condition, but which it is not yet diagnosed, (b) suppressing the disease or condition, i.e., stop its development, or (C) the weakening of the disease or condition, i.e. reverse the progression of the disease and the and condition, or alleviating one or more symptoms of a disease or condition. The group of patients being treated in this way, includes patient suffering from unwanted condition or disease, and patients at risk of developing a condition or disease. Thus a competent specialist in the field of technology suggests that treatment can improve the patient's condition, but may not completely cure the disease. As used in this context, the terms "violation" and "disease" are used interchangeably in regard to abnormal or pathological condition of the patient, which disrupts the body's functions and can be deadly.

The expression "therapeutically effective amount" is used in this context to denote a quantity sufficient to prevent, and preferably reduce by at least about 25%, more preferably at least 50%, most preferably at least 90% of clinically important changes in the nature of the pathology. In relation to the present invention, the term can also mean an amount sufficient to alleviate or reverse the development of one or more symptoms associated with a disease associated with deposition of amyloid proteins, such as Alzheimer's disease. In particular, terapeutiche the key effective amount" of a drug can result in relief, to reduce or eliminate at least one of the following symptoms: memory loss, constant sadness and anxiety, emptiness, hopelessness, pessimism, guilt, inadequacy, helplessness, loss of interest or pleasure in Hobbies and activities that once gave pleasure, low energy or fatigue, difficulty in concentrating, remembering, or making decisions; insomnia, early morning waking or excessive drowsiness, loss of appetite and/or weight, or overeating and weight gain thoughts of death or suicide and suicide attempts, restlessness, irritability and persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders and chronic pain. Applied to an individual active ingredient, administered as monotherapy, the term refers only to this ingredient. With regard to the combination, the term refers to combined amounts of the active ingredients that result in therapeutic effect, enter them in combination, sequentially or simultaneously.

The term "patient" refers to an animal, preferably a mammal, including Narimanov (e.g., cow, pig, horse, cat, dog, rat or mouse) and a Primate (e.g., obese the well or person). In a preferred embodiment, the patient is a dog. In a more preferred embodiment, the patient is a Primate, and in an even more preferred embodiment, the specified Primate is human.

In the context of the present invention, the expression "disease associated with deposition of amyloid proteins"includes diseases associated with the accumulation of amyloid protein, which can be either limited to a single organ, i.e., "localized amyloidosis"or spread to several organs, which is called "systemic amyloidosis". Disease associated with deposition of amyloid proteins, which can be treated by the compositions disclosed in this invention, include without limitation the disease are presented in table 1.

DiseaseTypical protein
Alzheimer's diseaseAmyloid-beta
Myositis with calves inclusions (IBM),Amyloid-beta
Diabetes mellitus type 2ARR (Amylin)
Parkinson's disease alpha synuclein
Transmissible spongiform encephalopathyPrion
Huntington's diseaseThe huntingtin
Medullary carcinoma of the thyroid glandCalcitonin
Heart arrhythmiaTrially natiure-
AtherosclerosisApolipoprotein AI
Rheumatoid arthritisSerum amyloid a And
Amyloid median aortaMedinas
ProlactinomaProlactin
Family amyloid polyneuropathyTransthyretin
Hereditary pneumopathiesLysozyme
Amyloidosis associated with dialysisβ2-microglobulin
Finnish amyloidosisGelsolin
Lattice degeneration of the corneaKeratoepithelin
Cerebral amyloid angiopathyAmyloid-beta
Cerebral amyloid angiopathyCystatin
Systemic AL amyloidosisLight chain

Secondary amyloidosis may be associated with chronic infection (such as tuberculosis) or chronic inflammation (such as rheumatoid arthritis), including family form of secondary amyloidosis, which see also in familial Mediterranean fever (FMF), and another type of systemic amyloidosis found in patients during long-time hemodialysis. Localized forms of amyloidosis include, without limitation, the type II diabetes and other related disorders, neurodegenerative diseases such as scrapie, bovine spongiform encephalopathy, a disease of Creutzfeldt-Jakob disease, Alzheimer's disease, cerebral amyloid angiopathy and disorders associated with pionowymi proteins. Sign amyloid disease is the deposition in the organs of amyloid plaques, composed primarily of fibrils, which, in turn, consist of characteristic proteins or peptides fibrils.

Consequently, in a specific embodiment, the disease associated with the deposition of melainah proteins, selected from Alzheimer's disease, disease of Creutzfeldt-Jakob disease, cerebral amyloid angiopathy and disorders associated with pionowymi proteins and muscle degenertive.

As will be borne in mind by a competent specialist, all specific embodiments of relating to the invention of a composition or kit, intended for use in medicine are also applicable when the above-mentioned composition or kit used for the treatment of diseases associated with deposition of amyloid proteins. As a consequence, specific embodiments of, relating to methods of treatment, corresponding to the invention, using a conjugate or composition, are:

a peptide selected from the C-terminal site /W-42), which is selected from Aβ(35-42) (SEQ ID NO:2), Aβ(33-42) (SEQ ID NO:3) and Aβ(33-40) (SEQ ID NO:4);

a peptide selected from the C-terminal site of Aβ(1-42), which

contains, in addition, an additional N-terminal Cys;

albumin, which is a bovine serum albumin

preferably, when the adjuvant is an adjuvant type Th1 or Th2, and

the composition of the invention or the active components of the set corresponding to the invention, which is injected once in two weeks.

A detailed description of these specific embodiments can be found in the preceding paragraph

In another specific embodiment, the disease associated with the deposition of amyloid protein selected from Alzheimer's disease, disease of Creutzfeldt-Jakob disease, cerebral amyloid angiopathy and disorders associated with pionowymi proteins.

The conjugate corresponding to the invention, or a composition corresponding to the invention, can be entered in various ways, for example intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, intradermally, orally, intranasally or intrabronchial, as well as topically, systemically or directly into the target (localized by). An overview of the different ways of administration of the active ingredients, fillers, designed for use, and methods of their preparation can be found in Tratado de Farmacia Galenica (Pharmaceutical reference Galanova drugs) C. Fault i Trillo, Luzan 5, S. A. de Ediciones, 1993 and Remington''s Pharmaceutical Sciences (Handbook of pharmaceutical Sciences, Remington (edited by A. R. Gennaro), 20 ed., Williams & Wilkins PA, USA (2000).

In the present invention, the expression "a localized way" is understood as the local introduction of the conjugate corresponding to the invention, in a specific area of the body of man or animal. Preferably, when the introduction of produce in the region with rapid blood flow, for example, intravenously in the peripheral or Central vein. Other ways may Nai and application, when the introduction is combined with how slow release or use of protective matrix. In addition, the appropriate way is through immunization through mucous nasal introduction, since it is known that this way of introducing favorable response of the Th2 type.

The dosing scheme is determined by the physician and clinical factors. As is well known in medicine, the dose depends on many factors, including physical characteristics of the patient (age, size, gender)used the method of administration, the severity of the disease, the specific connection and pharmacokinetic properties of the patient.

In a specific embodiment, the composition corresponding to the invention, or the active components of the set corresponding to the invention, is administered once every two weeks.

The composition conforming to the invention, or the active components of the set corresponding to the invention as shown efficient in terms of application in medicine.

For use in medicine, the composition conforming to the invention may be in the form of prodrugs, salts of MES, or clathrate, or in a dedicated form, or in combination with additional active agents. The composition corresponding to the present invention, it is possible to get together with a filler, which is acceptable from the point of view of the FA is Mazetti. Fillers, preferred for use in the present invention include sugars, starches, cellulose, gums and proteins.

As will be borne in mind by a competent individual, a composition corresponding to the invention, it is possible to get accepted methods known in the field of engineering, in solid (e.g. tablets, capsules, pills with sugar coating, granules, suppositories, crystalline or amorphous sterile solids, which can be recovered by obtaining liquid forms, etc), liquid (e.g., solutions, suspensions, emulsions, elixirs, lotions, ointments, etc.,) or semi-solid (gels, ointments, creams, etc.) pharmaceutical dosage form. Examples of pharmaceutically acceptable carriers known in the field of engineering and include phosphate buffered saline solutions, water, emulsions such as emulsions of oil/water, various types of wetting components, sterile solutions, etc.,

The following methods and examples should be interpreted as illustrative and not limiting the scope of invention.

Examples

Immunization of dogs C-terminal sequences of the beta-amyloid protein is safe and removes protein from the blood

Example 1. Immunization with peptides β(x-42)

I. Materials and methods

1. Characteristics of animals

The present invention uses twelve adult dogs on the odes Beagle both sexes. Animal characteristics are summarized in table 1. Animals obtained from commercial sources and include dog kennels, University of Santiago for cultivation. During the experiment animals contain three common rooms with free input/output and supply ready made food for dogs and water needs. Animals carefully identify immediately before the experiments, including physical and neurological examination, blood chemistry and complete blood count, and find that they are healthy.

Veterinarian, uninformed about treatment, and ultimately assigned to each group, distribute animals into four groups (a, b, C and D; n=3). Animals examined every week for the appearance of any clinical sign of reaction to the vaccine. For more complete examination, again including blood chemistry and complete blood count, carried out after the third immunization.

Animals are treated according to the European and Spanish law about the treatment of animals (86/609/EU, Real Decreto 1201/2005) and makes every effort to reduce the suffering of animals. The study was approved by the ethics Committee of the University of Santiago.

2. Getting immunogenic

Synthetic peptide AB(35-42) (with additional N-terminal Cys

when conjugation via SPDP) as antigen 0.4 mg/injection), included in four different vaccine preparation used for immunization of animals. Each of these drugs combined with protein carrier, or "blue media" (SU; Pierce, Rockford, IL, groups a and b)or bovine serum albumin (BSA; Sigma, Madrid, Spain; group D), and adjuvant or riverglen NDA (RH; Quimibios (manufacturer Reheis), Barcelona, Spain; groups a and C)or Abisco-300 (AB; Isconova AB, Uppsala Science Park, SE-751 83 Uppsala, SWEDEN; groups b and D).

2.1. Binding of synthetic peptides to BSA using SPDP:

First get the original solution of 20 mm SPDP (M-Succinimidyl 3-(2-pyridylthio)propionate, 50 mg/8 ml DMSO (dimethylsulfoxide)). Then 5 mg of BSA (bovine serum albumin) was dissolved in 1 ml of PBS (phosphate buffered saline)/5 mm EDTA (ethylenediaminetetraacetic acid), pH 8 BSA and added to 50 μl of a 20 mm solution of SPDP. The solution is incubated for 2 hours at room temperature. Then desalted column balance PBS/EDTA replace the buffer SPDP modified BSA to remove by-products of the reaction and the excess unreacted reagent SPDP.

Then determine the level of SPDP modification, using the following Protocol:

(a) 100 ál Diluted modified with SPDP and desalted BSA to 1 ml using PBS.

b) Measure and record the absorbance at a wavelength of 343 nm of the protein sample compared to a net analyst the PBS/EDTA (repeat test in three replicates).

c) Add 10 μl 15 mg/ml DTT (dithiothreitol) to a 1 ml sample of SPDP modified protein mix.

d) Exactly after 15 minutes, measure and record the absorbance at a wavelength of 343 nm of recovered sample.

e) Use the following equation:

When the value of X from 5 to 6.5 by 5 mg SPDP modified BSA add 2 mg of synthetic peptide.

After determining the level of SPDP-modified reaction mixture was incubated with stirring for 18-24 hours and lyophilizers the reaction mixture with the receipt of conjugates of bovine serum albumin - immunogenic peptide. For conjugates containing the immunogenic peptide β(35-42), Figure 1 shows the molecular weight distribution of the conjugates according to their average molecular weight.

2.2. Binding of synthetic peptides with the Blue carrier with the use of glutaraldehyde:

2 ml of borate buffer, pH 10 and 66 μl (200 mg/ml) Blue media added to the reaction container and gently mix. Then in the reaction capacity added 12 mg of synthetic peptide. Both glutaraldehyde diluted to 0.3% by adding borate buffer pH 10.

Then 1 ml of freshly prepared 0.3% solution of glutaraldehyde is added slowly into the reaction container under stirring at room temperature, allowing R is the action to proceed for 2 hours at room temperature in the dark (the solution becomes yellow). To block unreacted glutaraldehyde, the reaction capacity of add 250 µg 1M glycine and gently mixed incubare at room temperature for an additional 20 minutes.

After that the reaction mixture is transferred into a desalting column equilibrated PBS. The reaction mixture lyophilizer and diluted to a suitable concentration.

Thus, the present invention uses the following four

different drug:

A) 400 mcg Abeta35-42, conjugated with Blue media, with the addition of 200 ál of Revitagen NRA.

B) 400 mcg Abeta35-42, conjugated with Blue media, with the addition of 200 ál of Abisco-300.

C) 400 mcg Abeta35-42, conjugated to BSA, with the addition of 200 ál of Revitagen NRA.

D) 400 mcg Abeta35-42, conjugated to BSA, with the addition of 37 μl Abisco-300.

Used synthetic peptides represent:

When conjugation with Blue media by using glutaraldehyde:

1.β (35-42) (NH2-MVGGWIA-COOH) (SEQ ID NO:2) in dogs and mice, see below).

2. β (33-42) (NH2-GLMVGGWIA-COOH) (SEQ ID NO:3) in rabbits (see below).

When conjugation with BSA using SPDP:

1. Aβcys-(35-42) (NH2-CMVGGWIA-COOH) (SEQ ID NO:17) in dogs and mice, see below).

2. Aβcys-(33-42)(NH2-CGLMVGGVVIA-COOH) (SEQ ID NO:18) in rabbits (see below).

3. The immunization Protocol and sampling of blood

Animals (dogs) are divided into 4 groups before and will oznachaet for one of the four vaccines. Animals subjected to immunization Abeta35-42 subcutaneously in the back and monitorium adverse reactions. Scheme immunization and sampling is presented in Figure 2 (A). Briefly, animals do one injection every two weeks to seven times. Dogs in groups C and D receive an additional 8 immunization seven months after the seventh injection.

Blood samples collected from the jugular vein in polypropylene flasks with EDTA and protease inhibitors (complete minnebar mg/10 ml, Roche) immediately before the first immunization (W0 (week 0)and one week after the third week 5: W5), fifth (W9) and seventh (W13) immunizations (see Figure 2). Additional blood samples obtained from the animals of groups C and D four months after the seventh immunization (W31) and after one and three weeks after the booster immunization (W43 and W45, respectively).

Samples gently mixed and stored at 4°C for a maximum period of 2 hours before centrifugation at 4000 g for 10 minutes. Then take aliquots of plasma and frozen at -80°C until use. In addition, when each extraction ~1 ml of blood of each animal harvested in not containing EDTA polypropylene vial to obtain serum. The sample is allowed to form a clot for one hour at room temperature, then centrifuged at 4000 g for 10 minutes and collect the serum and stored at -80°C until analysis n the biochemical characteristics of blood.

Samples of cerebral spinal fluid (CSF) is collected under General anesthesia in aseptic conditions for one week prior to the first and at week 13 after the seventh immunization (see Figure 2). Take aliquots of these CSF samples and frozen at -80°C until use.

4. Analyses of antibodies to β in plasma and CSF

Antibodies to β determined by direct ELISA (ELISA test) in 96-well polypropylene plates. Holes for micrometrology cover 2.5 µg/ml human peptide β(1-42) (# 24224 AnaSpec. San Jose, CA, USA) in 100 mm sodium bicarbonate and 2M buffer on the basis of guanidine hydrochloride (pH 9,6) at 4°C over night. Then the tablets three times washed with 300 μl of buffer for washing (0.5 M Tris, 1.5 M sodium chloride, 0.5% tween-20, pH 8), block 300 μl of blocking buffer (0.05 M Tris, 0.2% tween-20 and 0.5% BSA; pH 8) for two hours at 37°C and washed again three more times. Tablets coated incubated for one hour at 37°C with 100 µl of the three-fold serial dilutions of samples of canine plasma in buffer medium (0.05 M Tris, 0.5 M sodium chloride, 0.05% of BSA, 0.05% tween-20; pH 8) in a row of 10 holes, starting with a dilution of plasma 1:30 in the first hole. The largest of the estimated dilutions of plasma is 1/10×310. The eleventh and twelfth columns in each tablet fill buffer carrier without plasma as blank controls. Then the tablets wash the Ute and incubated for one hour at 37°C with 100 µl dilution 1:1000 in buffer media horseradish peroxidase, conjugated with rabbit IgG to the dog (Jackson InmunoResearch. Suffolk, UK), washed three times and incubated with 0,0375% ABTS (Roche, Barcelona, Spain) in the buffer for ABTS (Roche. Barcelona, Spain). The absorption at a wavelength of 405 nm is read on an automated plate reader for tablets (Synergy 4, Biotek. Winooski, Vermont, USA).

The concentration of antibodies to Aβ in plasma calculated using monoclonal antibody E as standard on the same tablets for ELISA and expressed in μg/ml Is AS for each plasma sample is determined by nonlinear regression absorption relative to the logarithm of the dilution in each well (GraphPad Prism 3.02). In addition, the final titer in plasma is defined as the maximum dilution of plasma, in which the absorption is three times greater than the average absorption of empty holes.

The definition of free Aβ peptides in plasma

The levels of Aβ(1-42) and Aβ(1-40) in plasma and CSF of dogs is measured using an indirect sandwich ELISA using kits ELISA ABtest-40 and ABtest-42 company Araclon Biotech (Zaragoza, Spain), following the manufacturer's instructions.

II. Results

Animals remain healthy and active throughout the entire period of the experiment. In particular, do not reveal any signs of a reaction to the vaccine. The average increase in body weight from week 0 to week 13 is 1±1.7 kg (see table 1). Only two animals decreased body weight during this period (1 kg for each is, that is 4% and 7% of their weight at week 0, respectively.

Table 1. Characteristics of animals
Group/DogFloorAge (years)Mass (kg) WMass (kg) WThe increase in the mass (kg)Weight change, %
A1female111618212,50
A2male62322-1-4,35
A3male82223,451,456,59
B1female1013130000
B2male6212329,52
B3male1224,4526,72,259,20
C1female6181800,00
C2male6161600,00
C3male1024,5294,518,37
D1male61413-1-7,14
D2 male6151500,00
D3male1018,7a 21.52,814,97
Average18,8019,891,08equal to 4.97
SD*4,105,221,688,03
* standard error

The titers of antibodies to β(1-42)

Groups a and b

Interpolation of the values of the absorption in the plasma of animals in group a and In a relatively standard E does not detect a systematic difference between preimmune samples and samples collected at weeks W5, W9 W13 (see Table 2A and Figure 3A). Procedure increment in antibody titers in plasma, as measured equivalence number µg/µl E, after three and seven immunizations (W5 and W13, respectively) is very low (0,8±0,2 and 0,8±0,2 respectively for group A; 1,2±0,1 and 1.3 ą 0.3, respectively, for group C. Cm. table 3). The values of EC50plasma for specific antibodies (β42) in animals of groups a and b are very low from week 0 to week 13. In fact, the values of EC50plasma reduced from W0 to W5 all data dogs except dogs B2, which shows a negligible increment exclusively at this point of time (see table 2B and Figure 3A-C). In addition, even the most concentrated dilution of plasma (1:30) data dogs after immunization gives the absorption values three times higher than this value, measured in samples preimmune plasma (Table 2C). So consider that dogs in these two groups do not respond to appropriate vaccine preparations (a and b).

Table 2. Titers in the plasma of specific antibodies against Aβ42 in different points of time
Panel A: equivalent μg/ml of monoclonal antibodies E
Group/DogW 5W 9W 13W 31W 43W 45
A10,00280,00160,00150,0016
A20,00240,00230,00180,0023
A30,00270,00220,00280,0021
B10,00190,00230,00700,0033
B20,00250,00310,00220,0028
B30,00270,00280,00290,0032
C10,00160,02050,01470,00840,00080,00170,0019
C20,00100,01130,00830,00930,00070,00400,0041
C30,00220,02760,214 0,02060,00210,00890,0102
D10,00250,10750,09330,10480,00760,07160,0520
D20,00170,05290,04060,02940,00280,02290,0164
D30,00190,03210,01380,02560,00240,00880,0071
Panel: values AS plasma for specific antibodies (β42)
A149423529
A217085158133
A3155111132115
B1514723566
B218625312393
OT79604159
C145773722385684649
C214747335038570114129
NW1231349 99485370336460
D15167595738630231942022733
D253295620211378661122790
D3571550752116961365267
Panel: the reciprocal of the maximum dilution of the plasma with the absorption of 3
A127027090270
A2270270270270
A3270270270270
B1270270810810
B2270810270270
OT270270270270
C190243024302430270270270
C29024302430243090810810
NW27024302430243081024302430
D12702187021870218702430218707290
D227072907290729081072902430
D32707290243072908102430810

C1
Table 3. Paradegermany relatively preimmune plasma (IFF
Group/DogW0/W0W5/W0W9/W0W13/W0W31/W0W43/W0W45/W0
A11,00,60,50,6
A21,01,00,81,0
A31,00,81,00,8
Average1,0±0,00,8±0,2 0,8±0,30,8±0,2
B11,01,23,61,7
B21,01,20,91,1
B31,01,11,11,2
Average1,0±0,01,2±0,11,9±1,51,3±0,3
1,012,58,95,10,51,01,1
C21,011,88,6the 9.70,74,24,2
C31,012,6the 9.79,41,04,14,6
Average1,0±0,012,3±0,49,1±0,68,1±2,60,7±0,23,1±1,83.3V±1,9
D11,042,837,2 41,73,028,520,7
D21,030,623,5of 17.01,613,29,5
D31,017,17,413,71,3the 4.7the 3.8
Average1,0±0,030,2±12,822,7±14,24,1±15,32,0±0,915,5±12,111,3±8,6

Groups C and D

In contrast to that observed in groups a and b in dogs treated with vaccine preparations C and D, shows a significant modification of the antibody titers in the plasma. Answer all the dogs in these two groups, as measured by the standard E, and EC50or end the breeding of the same type, although quantitative differences observed both between and within groups (see Table 2A-C). The primary type is characterized by a significant increase in itrow after three immunizations, preserved with only minor deviations from W5 to W13 (see Figures 3A-C). Thus, the antibody titers significantly increased after three immunizations, but not increase substantially (or even fall slightly) after four subsequent injections of the vaccine, held once in two weeks. Specifically titers, as measured equivalence number µg/ml E, after three and seven immunization (W5 and W13, respectively) multiplied by the order of 12.3±0,4 and 8.1±2,6 respectively for the group; 30,2±12,8 and 24.1±15,3, respectively, for group D (see Table 3).

Four months after the last immunization (W31) titles fall to very low levels (see table 2). However, in dogs of group D, they are still two times higher than in preimmune plasma (see table 3). Then the dog groups C and D receive an additional eighth immunization carried out seven months after the seventh appropriate vaccine preparation. After this booster injections should a significant increase in antibody titers in group D (increase of 15.5±12,1 times on preimmune plasma, see table 3) and, to a lesser extent in the group (increase of 3.1±1.8 relatively preimmune plasma, see table 3) (see Figures 3A-C). Typically, at any point in time the antibody titers are always higher in the animals of group D than in animals of group C. However, the increment t the TRS in dogs with a lower response in group D (case D3) is very close to the increments of titles in dogs in group C (see table 3, figures 2, 3).

2. The title peptide β

Sandwich ELISA does not detect significant differences in concentrations of peptides β1-42 or β1-40 between preimmune plasma samples collected in week W5, W9 and W13 animals areagirls groups (a and b) (see table 4A, Figure 4A-C). The order of concentration change β1-42 in plasma after three and seven immunization (W5 and W13, respectively) are very low (1.2±0,7 and 1,0±0,0 respectively for group A; 0,9±0.0 and 1.0±0.1, respectively for group C. Cm. table 5A). Similarly, the procedure for changing the concentration of Aβ1-40 in plasma after three and seven immunization (W5 and W13, respectively) are very low (0,9±0,1 for two weeks in both groups. Table 5B).

Table 4A. The concentration of the peptide Aβ(1-42) in PG/ml
Group/DogW 0W 5W 9W 13W 31W 43W 45CSF WCSF W
A14,167,50was 2.76or 4.31 556,85509,70
A232,9232,9232,9232,71253,06322,77
A319,9820,0720,2419,62421,88300,12
B147,0046,4047,0047,00299,86420,84
B220,3320,3321,8322,01283,94367,18
OT 33,3333,3332,8232,71314,82228,06
C15,20<3,125<3,125<3,1254,70<3,125<3,125718,84532,56
C226,4615,00<3,125<3,12517,53<3,125<3,125519,74581,28
NW16,35<3,125<3,125<3,125<3,125<3,125<3,125671,03649,07
D174,7630,583,33 28,6041,9642,6229,10677,44731,28
D217,56<3,125<3,125<3,125<3,125<3,125<3,125783,95888,61
D316,8515,51<3,125<3,125<3,125<3,125<3,125488,97762,05

Table 4B. The concentration of the Aβ peptide(1-40) in PG/ml
Group/Dog W 0W 5W 9W 13W 31W 43W 45CSF WCSF W
A161,2952,5552,5555,193633,93831,7
A258,6752,7655,2160,842269,62719,6
A360,2664,8863,0054,642534,52822,0
B1104,3138,7039,86<3,125 2890,53586,1
8268,7758,6763,8765,162371,52610,9
OT79,3080,8993,8784,782449,42273,4
C127,47<3,12521,72<3,12524,44<3,125<3,1253503,82711,6
C256,8933,0329,6235,5339,3928,4921,672632,5 3416,4
C341,67<3,125<3,125<3,12527,80<3,125<3,1253121,22945,8
D1106,9459,0648,6946,1470,7146,7855,073139,03206,7
D259,70<3,125<3,12522,0424,92<3,12525,553737,73426,4
D351,6738,7126,44<3,125of 33.26<3,12535,302955,13214,8

Table 5A. The order of the increments of the peptide β (1-42)
Group/DogW0/W0W5/W0W9/W0W13/W0W31/W0W43/W0W45/W0
A11,01,80,71,0
A21,01,01,01,0
A31,01,01,01,0
mean±1,0±0,01,2±0,50,8±0,21,0±0,0
B11,01,01,01,0
B21,01,01,11,1
B31,01,01,01,0
mean±1,0±0,00,9±0.01,0±0.01,0±0.1
C11,00,20,20,20,90,20,2
C21,00,60,00,0 0,70,00,0
C31,00,10,10,10,10,10,1
mean±1,0±0,00,2+0,30,0±0,10,0±0,10,5±0,10,0±0,10,0±0,1
D11,00,40,30,40,60,60,4
D21,00,10,10,10,10,10,1
D31,00,90,10,10,10,10,1
mean±1,0±0,00,4±0,40,1±0,10,1±0,20,2±0,30,2±0,30,1±0,2

0,4
Table 5B. Order increment peptide β (1-40) with respect to preimmune
Group/DogW0/W0W5/W0W9/W0W13/W0W31/W0W43/W0W45/W0
A11,00,90,90,9
A21,00,90,91,0
A31,01,11,00,9
mean±1,0±0,00,9±0,10,9±0,10,9±0,1
B11,01,01,01,0
B21,00,90,90,9
B31,01,01,21,1
mean±1,0±0,0 0,9±0,11,0±0,10,9±0,1
C11,00,00,80,00,90,00,0
C21,00,60,50,60,70,50,4
C31,00,00,00,00,70,00,0
mean±1,0±0,00,2±0,30,4±0,40,2±0,30,7±0,10,1±0,30,1±0,2
D11,00,60,50,40,70,5
D21,00,00,00,40,40,00,4
D31,00,70,50,00,60,00,7
mean±1,0±0,00,4±0,40,3±0,30,2±0,20,5±0,10,1±0,20,5±0,1

In contrast to that observed in groups a and b, in dogs treated with vaccine preparations C and D, show a significant modification of the concentrations β(1-42) in the plasma. This change corresponds to the type characterized by a significant decrease in the level β(1-42) in the week W5, after three immunizations, to the point where five of the six dogs peptide becomes undetectable (<3,125 PG/ml, see table 4A, Figure 4A). The concentration of the peptide is maintained at undetectable levels until week W13, in the experiment increased by W31, through catherinezeta after the seventh immunization, when the antibody titers are approaching preimmune levels, and then decreases again after the booster (8) injection at week W42 (see Figure 2). The magnitude of these changes is presented in Table 5A. Somewhat unexpectedly, the concentration of β1-40 plasma, apparently, corresponds to the type of change, very close β(1-42) (see tables 4B and 5B, Figure 4B). According antibody titers and concentrations of the peptide at any point in time represent in Figure 5.

Despite significant differences between groups C and D in terms of antibody titers in plasma at any time point during the experiment, fluctuations of concentrations of Aβ peptide occur in the same period of time. This suggests that even high titers of antibodies is not sufficient to achieve removal of Aβ peptides from the bloodstream.

Example 2: Immunization with peptide β(x-40) I. Materials and methods

1. Characteristics of animals

In this invention using six adult dogs breed Beagle both sexes. Animals obtained from commercial sources and include dog kennels, University of Santiago for cultivation. During the experiment animals contain three common rooms with free input/output and supply ready made food for dogs and water needs. Animals carefully identify immediately before the experiment is in, including physical and neurological examination, blood chemistry and complete blood count, and find that they are healthy.

Veterinarian, uninformed about treatment, and ultimately assigned to each group, distribute animals into two (a and b, n=3). Animals examined every week for the appearance of any clinical sign of reaction to the vaccine. For more complete examination, again including blood chemistry and complete blood count, carried out after the third immunization.

Animals are treated according to the European and Spanish law about the treatment of animals (86/609/EU, Real Decreto 1201/2005) and makes every effort to reduce the suffering of animals. The study was approved by the ethics Committee of Universiteta Santiago.

2. Getting immunogenic

Synthetic peptide AB(33-40) (with additional N-terminal Cys at the conjugation by SPDP) as antigen (0.4 mg/injection)are included in two different vaccine preparation used for immunization of animals. Each of these drugs combined with protein carrier, or "blue media" (SU; Pierce, Rockford, IL, group a)or bovine serum albumin (BSA; Sigma, Madrid, Spain; group b), and adjuvant Abisco-300 (AB; Isconova AB, Uppsala Science Park, SE-751 83 Uppsala, SWEDEN).

For the conjugation of peptides containing N-terminal cysteine, Blue carrier is albumin using SPDP use the same method, that in Example 1 (see section 2.1 Materials and methods).

In this invention using the following two different drug:

(A) 400 mcg of synthetic peptide conjugated to Blue media, with the addition of 200 ál of Abisco-300.

(B) 400 mcg of synthetic peptide conjugated to BSA, with the addition of 37 μl Abisco-300.

Used synthetic peptides represent:

When SPDP conjugation with:

ABcys-(33-40) (NHa-CGLMVGGW-COOH) (SEQ ID NO:19) in rabbits (see below).

3. The immunization Protocol and sampling of blood

Animals are divided into 2 groups and determine one of the two vaccines (see. above (a) and (b)). Animals subjected to immunization subcutaneously in the back and monitorium adverse reactions. Scheme immunization and sampling is presented in Figure 2 (B). Briefly, animals inject once in two weeks seven times.

Blood samples collected from the jugular vein in polypropylene flasks with EDTA and protease inhibitors (complete minnebar mg/10 ml, Roche) immediately before the first immunization (W0 (week 0)and week after that (W1), week 5 (W5), week 7 (W7), week 9 (W9), week 11 (W11 and at week 13 (W13) after the first immunization (see Figure 2 (C)).

Samples gently mixed and stored at 4°C for a maximum period of 2 hours before centrifugation at 4000 g for 10 minutes. Then take aliquots of plasma and frozen at -80 the C before use. In addition, when each extraction ~1 ml of blood of each animal harvested in not containing EDTA polypropylene vial to obtain serum. The sample is allowed to form a clot for one hour at room temperature, then centrifuged at 4000 g for 10 minutes and collect the serum and stored at -80°C until analyzed at the biochemical characteristics of blood.

Samples of cerebral spinal fluid (CSF) is collected under General anesthesia in aseptic conditions for one week prior to the first (W0) and at week 13 (W13) after the seventh immunization (see Figure 2 (B)). Take aliquots of these CSF samples and frozen at -80°C until use.

4. Analyses of antibodies to β in plasma and CSF

Antibodies to β determined by direct ELISA in 96-well polypropylene plates. Holes for micrometrology cover 2.5 µg/ml human peptide W-40) (# 24224 AnaSpec. San Jose, CA, USA) in 100 mm sodium bicarbonate and 2M buffer on the basis of guanidine hydrochloride (pH 9,6) at 4°C over night. Then the tablets three times washed with 300 μl of buffer for washing (0.5 M Tris, 1.5 M sodium chloride, 0.5% tween-20, pH 8), block 300 μl of blocking buffer (0.05 M Tris, 0.2% tween-20 and 0.5% BSA; pH 8) for two hours at 37°C and washed again three more times. Tablets coated incubated for one hour at 37°C with 100 µl of a threefold serial dilution of samples dog plasmas is in the buffer medium (0.05 M Tris, 0.5 M sodium chloride, 0.05% of BSA, 0.05% tween-20; pH 8) in a row of 10 holes, starting with a dilution of plasma 1:30 in the first hole. The largest of the estimated dilution of plasma is 1/10x310. The eleventh and twelfth columns in each tablet fill buffer carrier without plasma as blank controls. Then the tablets are washed and incubated for one hour at 37°C with 100 µl dilution 1:1000 in buffer media of horseradish peroxidase conjugated with rabbit IgG to the dog (Jackson InmunoResearch. Suffolk, UK), washed three times and incubated with 0,0375% ABTS (Roche, Barcelona, Spain) in the buffer for ABTS (Roche. Barcelona, Spain). The absorption at a wavelength of 405 nm is read on an automated plate reader for tablets (Synergy 4, Biotek. Winooski, Vermont, USA).

The concentration of antibodies to Aβ in plasma calculated using monoclonal antibody E as standard on the same tablets for ELISA and expressed in μg/ml Is AS for each plasma sample is determined by nonlinear regression absorption relative to the logarithm of the dilution in each well (GraphPad Prism 3.02). In addition, the final titer in plasma is defined as the maximum dilution of plasma, in which the absorption is three times greater than the average absorption of empty holes.

The definition of free peptides β plasma

Levels β(1-42) and β(1-40) in plasma and CSF of dogs is measured using an indirect sandwich ELISA with the use of what Finance kits ELISA ABtest-40 and ABtest-42 company Araclon Biotech (Zaragoza, Spain), following the manufacturer's instructions.

II. Results

Animals remain healthy and active throughout the entire period of the experiment. In particular, do not reveal any signs of a reaction to the vaccine.

1. The titers of antibodies to β(1-40)

Dog treat vaccine drug more effectively, demonstrating the substantial modification of antibody titers to β40 in their plasma. Answer all the dogs in this group, as measured by the concentration of antibodies to β40, and EC50or end-breeding, correspond to one and the same type, although quantitative differences observed both between and within groups (see Table 6, Panels a-b). The primary type of dogs treated with a drug, characterized by a significant increase in titers after two immunizations (W5), which are stored only small deviations from W5 to W9 (see Figure 6A). Thus, the antibody titers significantly higher after two immunizations, but not increase or even fall slightly after subsequent injections of the vaccine, in particular, after the seventh immunization (W13) titles a little fall to levels lower than the values W5 (see Table 6 and Figure 6A) in group C.

Table 6. The titers of specific antibodies to β40 in plasma at various points
Panel A: Equivalent to the number of ng/ál antibody β40
Group/DogW0W5W9W13
A10,34830,47050,72510,8830
A20,41960,58760,62840,4501
A30,52140,44500,52140,4603
B10,358510,186011,67298,0932
B20,79137,13599,93146,0361
B31,61112,22722,26292,5175
Panel: values AS plasma for specific antibodies (β40)
A1 49,4726,9238,5431,01
A2BUTBUTBUT14,97
A325,229,8424,1446,67
B131,4413281593831,1
B266,85906,81031571,9
B342,15150,9154129,1
BUT: not defined

2. The title peptide Aβ

In dogs treated with vaccine drug To show significant modification concentrations β(1-42) in the plasma. These changes correspond to the type characterized by a significant decrease in the level of peptide β(1-42) in the week W5, after two immunizations, to the point where the peptide becomes undetectable (<3,125 PG/ml; see Table 7A).

The concentration of the PE the Chida remains undetectable until week W13. However, in dogs treated with the vaccine with the drug And did not show any changes in concentrations of Aβ(1-42) in the plasma. The concentration of the Aβ peptide(1-40) in plasma, appears to be consistent with the type of changes that are very close to Aβ(1-42) (see table 7).

After comparing the levels of CSF prior to immunization (W0) and at week W13 using ELISA shows that the levels of peptide β(1-40) and Aβ(1-42) is decreased in animals of both groups (a and b) after treatment with the vaccine. Suddenly it turns out that the levels are even lower in dogs from group a than in dogs from group b, although the dogs from group a did not show immunogenic response.

Table 7A. The concentration of the peptide Aβ(1-42) in PG/ml
Group/DogW0W1W5W7W9W11W13CSFCSF
A1<3,125<3,125<3,125<3,12513,2812,0013,60464,3544177
A2at 10.6411,4913,1910,0815,17br12.6215,17349,84160,32
A317,1514,6014,0414,6015,1718,8515,74345,00196,61
B117,4323,81<3,125<3,125<3,125<3,125<3,125403,06237,74
B230,1918,38<3,125<3,125<3,125<3,125<3,125263,55195,81
B3<3,12 12,34<3,125<3,125<3,125<3,125<3,125261,13200,65

Table 7B. The concentration of peptide β(1-40) in PG/ml
Group/DogW0W1W5W7W9W11W13CSF W0CSF W13
A147,8947,7049,9549,0150,1452,0154,642576,972053,48
A248,5647,3640,1640,7642,3645,5648,362734,55 1414,09
A337,7644,9640,3645,3650,7642,7649,162616,361393,64
B159,3351,08<3,125<3,125<3,125<3,125<3,1252752,732363,33
B267,2052,20<3,125<3,125<3,125<3,125<3,1251665,611042,88
B346,3644,56<3,125<3,125<3,125<3,125<3,1251831,521676,97

Example 3: Effect of immunization is the brain

I. Materials and methods

To study the effect of immunization in the brain carry out acute experiment, to which animals are put to death after three immunizations with the following medications:

(1) Aβ X-42+BSA+adjuvant Th2 dysbalance-type (Rapidreel).

(2) Aβ X-42+BSA+adjuvant mixed Th1/Th2 dysbalance-type (Abisco).

(3) Control: albumin+adjuvant Th2 dysbalance-type.

Samples of the frontal, entorhinal, temporal and cerebellar regions of the brain homogenized in TBS, pH 7.4, containing inhibitor cocktail protease, followed by centrifugation at 175000 g for 30 minutes at 4°C, using the device Beckman MLA-55. The resulting supernatant is considered soluble fraction of each of them. Then again homogenized sediment in TBS-TX pH 7.4, containing inhibitor cocktail protease, and centrifuged in the same conditions as previously. Again the resulting precipitate is re-homogenized in TBS-guanidinate and incubated over night at room temperature while rotating the mixer, followed by centrifugation at 13,000 g for 1 hour 30 minutes at 4°C, using the device Beckman MLA-55. The precipitate discarded and the resulting supernatant resuspended in TBS-guanidinate and consider the insoluble fraction. Then the concentration of peptides quantitatively determined using ELISA.

II. Results

Immunization with Aβ X-42, conjugated with BSA, d is et in the strong effect of soluble forms of the peptides at lower concentrations of more than 50% in each treatment group relative to the control group. The concentration of insoluble forms substantially not reduced (see Figure 7). About different areas of the brain, decreased similarly in all areas for the soluble forms of the peptides β40 and β42 minor differences (see Figure 8).

As shown in Examples 2 and 3, differences in the concentration of peptide in the CSF are missing compared to the original level and time point after treatment.

The results obtained in the present invention, consistent with the advanced idea that immunization β should be more effective if it is conducted before began aggregation β.

1. The conjugate containing 1÷93 immunogenic peptides β(35-42) (SEQ ID NO: 2), or 1÷93 immunogenic peptides β(33-42) (SEQ ID NO: 3), or 1÷93 immunogenic peptides β(33-40) (SEQ ID NO: 4), as well as the native albumin, while peptides bound to albumin linker section containing cysteine and bifunctional linker, and the linker section attached cysteine to the N-end is not more than one immunogenic peptide for the treatment or prevention of diseases associated with the deposition of amyloid proteins.

2. Application under item 1, in which the disease associated with the deposition of amyloid protein selected from the group of diseases, including Alzheimer's disease, Creutzfeldt-Jakob disease, cerebral amyloid of Angi is patio and violations associated with pionowymi proteins.

3. Application under item 1, in which native albumin is a bovine serum albumin.

4. Application under item 1, in which the conjugate is administered once every two weeks.

5. The use of a composition containing an effective amount of a conjugate consisting of 1÷93 immunogenic peptides Aβ(35-42) (SEQ ID NO: 2), or 1÷93 immunogenic peptides β(33-42) (SEQ ID NO: 3), or 1÷93 immunogenic peptides β(33-40) (SEQ ID NO: 4), as well as the native albumin, while peptides bound to albumin linker section containing cysteine and bifunctional linker, and the linker section attached cysteine to the N-end is not more than one immunogenic peptide and adjuvant for the treatment or prevention of a disease associated with deposition of amyloid proteins.

6. Application under item 5, in which the adjuvant is an adjuvant selected from the group comprising adjuvant type Th1 adjuvant type and Th2 adjuvant mixed type Th1/Th2.

7. Application under item 5, in which the disease associated with the deposition of amyloid protein selected from the group of diseases, including Alzheimer's disease, Creutzfeldt-Jakob disease, cerebral amyloid angiopathy and disorders associated with pionowymi proteins.

8. Application under item 5, in which native albumin is a bovine serum albumin.

9. Applied the e on p. 5, in which the composition is administered once every two weeks.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula I, possessing a modulating action with respect to the CC chemokine receptor 3 (CCR3), a based on them pharmaceutical composition, versions of treatment methods and a method of controlling the CCR3 activity. In the general formula I R1 and R2 represent halogen or C1-6alkyl; R3 represents cyano or nitro; R4 represents or ; R5 represents oxo; C1-6alkyl, optionally substituted with halogen atoms; or C(O)OR1a; X represents O or S; Y represents -O-, -S-, -N(R1a)-, -C(R1a)(R1d)- or -C(R1a)(NR1bR1c)-; m represents an integer number from 0 to 2; n represents 1; p represents an integer number from 0 to 2; r represents 1 or 2; and each R1a, R1b, R1c and R1d represents (a) hydrogen; (b) C3-7cycloalkyl; or (c) C1-6alkyl, optionally substituted with hydroxyl, or each pair R1b and R1c together with a N atom, which they are bound to, form imidazoimidazolyl, substituted with oxo, butyl or chlorine, or heterocycle, containing 5 or 6 atoms in a cycle.

EFFECT: improvement of the composition properties.

41 cl, 2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to bi- and polycyclic substituted isoquinoline and isoquinolinones of formula (I), or to its stereoisomeric and/or tautomeric forms and/or to its pharmaceutically acceptable salts, where R1 represents OH; R3, R4, R5 and R8 represents H; R7 represents halogen or (C1-C6) alkyl; R6 represents one (C1-C4)alkylene, bound to a cycloalkyl ring, in which (C1-C4)alkylene forms the second bond with the other carbon atom of the cycloalkyl ring with the formation of a bicyclic ring system, where in the bicyclic ring system one carbon atom is substituted with a group, independently selected from O, S or SO2; and if m and s equal 2 or m equals 3 and s equals 1, R6 represents a group CH2-CH-(CH2)2, which via one group CH2 is bound to the cycloalkyl ring, and two other CH2 groups are bound to different carbon atoms of the cycloalkyl ring, and if m equals 3 and s equals 3, R6 represents two methylene groups, bound to different carbon atoms of the cycloalkyl ring, where the methylene groups or group CH2-CH-(CH2)2 are bound to the carbon atoms of the cycloalkyl ring and form an adamantane system of formula (XX) , where L can be bound to any secondary or tertiary carbon atom, or R6 together with R11 and an N atom form (C5) heterocycloalkyl, bound with the cycloalkyl residue in the form of a spirocyclic ring system, where the bicyclic ring system, or the adamantane system, or a ring system, containing (C5) heterocycloalkyl, represent non-substituted or optionally substituted with substituent R9; R9 represents (C1-C6)alkyl, (C2-C6)alkenyl, (C6)aryl or cyclopropyl R11 and R12 independently on each other represent H or (C1-C6)alkylene-(C6)aryl; n equals 0 or 1; m equals 2 or 3; s equals 1, 2 or 3; L represents O; its stereoisomeric and/or tautomeric forms and/or its pharmaceutically acceptable salts. The invention also relates to the application of a formula (I) compound.

EFFECT: novel bi- and polycyclic isoquinoline and isoquinolinone derivatives, useful as inhibitors of Rho-kinase, are obtained.

22 cl, 22 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the application of isorhamnetin triglycosides of general formula 1 , where R1=α-L-rha, R2=β-D-xyl, (compound 2), or where R1=α-L-rha-(1->4)-α-L-rha, R2=H, (compound 3), or where R1=α-L-rha, R2=β-D-xyl, (compound 4) and extracts, containing the said compounds, for treatment or prevention of neurological or psychic diseases, associated with the reduction of the everyday activity and/or associated with the impairment of mental functions, influencing the everyday activity, and/or with the impairment of attentiveness, and a condition of excitation, selected from the attention deficit hyperactivity disorder (ADHD), chronic fatigue syndrome, state of exhaustion, dementia, Alzheimer's disease, vascular dementia, memory impairment caused by other reasons, and for memory improvement. In addition, the claimed invention relates to triglycoside of general formula I, representing compound 2.

EFFECT: increased efficiency of the compound application.

25 cl, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: group relates to peptides or polypeptides inducing the anti-alpha-synuclein antibody production in vivo for producing medicaments for preventing and/or treating synucleinopathies.

EFFECT: producing the peptides or polypeptides, which induce the antibodies responsible for the removal of the alpha-synuclein involved in the production of alpha-synuclein aggregates, the Lewy bodies, or for the dissolution of alpha-synuclein aggregates, the Lewy bodies in the individual suffering from synucleinopathies.

22 cl, 3 ex, 5 tbl, 5 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a compound presented by formula (E) , wherein X, Y and L are independently non-directionally specified in -C(R1)(R2)-, -C(R3)=, -N(R4)-, -N= and -O-; M and Z are independently non-directionally specified in ; ---- means an optional double bond; R1, R2, R3, R4 and R6 are independently specified in hydrogen; C1-4 alkyl; group -C1-4 alkylene-halogen; group -C1-4 alkylene-OH; Hal is specified in F, Cl, Br and I; RE1 and RE2 are attached to neighbouring carbon atoms, and RE1 and RE2 together non-directionally form the structure -T-(CRE7RE8)n-V-, wherein T is specified in CRE9RE10 and O or NH, and V is specified in CRE9RE10 and O or NH, as well as respective structures comprising a double bond; at least one of T or V represents O or N; RE7 and RE8 represent H or F; RE9 and RE10 represent H; n takes on the values of 1 to 2; RE3 represents C1-6 alkyl group; m takes on the values of 0 or 1; RE4 represents a halogen atom; p takes on the values of 0 or 1; as well as to pharmaceutical diagnostic compositions of the above compound.

EFFECT: preparing the new pharmaceutical compounds.

41 cl, 17 dwg, 2 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula I: cis-COOR-XCH-(CH2)a-CH=CH-(CH2)b-CH3, wherein (a) and (b) can take any value from 0 to 14, (X) is specified in: OH, NH2, CH3, F, F3C, HS, O-CH3, PO4(CH2-CH3)2 and CH3COO, and (R) represents sodium (Na) applicable for preventing and/or treating obesity, hypertension and/or cancer. Also, the invention refers to using the compounds of formula I for preparing a pharmaceutical and/or nutrient composition, to the pharmaceutical and/or nutrient composition based on the compounds of formula I, to a cosmetic, non-therapeutic method for improving skin manifestations and to a method for preventing and/or treating the diseases in humans and animals with using the compounds of formula I.

EFFECT: preparing the new compounds.

18 cl, 22 dwg, 5 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: claimed group of inventions relates to medicine, namely to neurology, and deals with treatment of organic diseases of the nervous system, including psychoorganic syndrome and encephalopathies of different genesis. For this purpose a pharmaceutical composition, which contains an activated potentiated form of antibodies to brain-specific protein S-100 and an activated potentiated form of antibodies to endothelial NO-synthase, is introduced.

EFFECT: invention provides efficient treatment of organic disease of the nervous system due to the synergic action of the composition components.

11 cl, 5 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: invention concerns the pharmaceutical composition Laquinimod (5-chloro-N-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide) in the free form and/or in the form of a pharmaceutically acceptable salt in making a finished pharmaceutical product for treating and/or relieving, and/or delaying the clinical course and progression of multiple sclerosis. As excipients, the composition contains lactose, starch and/or starch derivatives, gelatine, talc and/or paraffin.

EFFECT: invention enables extending the range of high-bioavailability products for treating multiple sclerosis.

3 cl, 5 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to medicine, namely to therapy, and deals with treatment of lysosomal storage disease - Niemann-Pick A or B disease. For this purpose enzyme acid sphingometlinaise is slowly, for three hours, introduced intraventricularly.

EFFECT: method and mode of introduction provide effective treatment of said disease due to reduction of pathological level of acid sphingomyelinase substrate both in tissues of brain and in visceral organs.

9 cl, 13 dwg, 5 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to nutritive therapy and is intended for treatment and/or prevention of delayed recall function impairment in subjects who have 24-26 points by Mini-mental State Examination (MMSE). Claimed is application of composition for obtaining product for enteral introduction. Composition contains uridine or uridine phosphate, docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA), phospholipids, choline, vitamin E, vitamin C, selenium, vitamin B12, vitamin B6 and folic acid.

EFFECT: claimed invention provides improvement of memory, in particular, delayed recall function, in patients with 24-26 points by MMSE.

5 cl, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of conjugation of polysialic acid (PSA) or modified PSA (mPSA) with an oxidised carbohydrate part of glycoprotein, different from the blood clotting protein, which contains a carbohydrate group, by contact of the oxidised carbohydrate part with PSA or mPSA, where the said PSA or mPSA contains an aminooxygroup and an oxime bond is formed between the oxidised carbohydrate part and the aminooxygroup on PSA or mPSA.

EFFECT: method makes it possible to obtain high conjugate output, high preservation of the conjugated glycoprotein activity in comparison with the non-conjugated protein and high conjugation effectiveness.

14 cl, 2 dwg, 27 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a metal-salen complex derivative. The complex is represented as A-B-C, where A is the metal-salen complex, B is a bond zone, including at least one disulphide bond; and C is a functional molecule, consisting of at least one of enzymes, antibodies, antigens, peptides, amino acids, oliginucleotides, proteins, nucleic acids and medication molecules. The bond zone (B) includes a molecule of a cross-linking agent to form a cross-linking between the said metal-salen complex (A) and the said functional molecule (C). The said metal-salen complex (A) and the said molecule of the cross-linking agent are bound together via at least one disulphide bond; and the said molecule of the cross-linking agent and the said functional molecule (C) are bound together via at least one disulphide bond. The zone of the disulphide bond (B) results from the formation of a bond between a SH group, introduced as a substituent into the said metal-salen complex, and the SH group of the said functional molecule (C), or results from the formation of a bond between the SH group of the said metal-salen complex (A) or the said functional molecule (C) and the SH group of the cross-linking agent molecule. Also claimed is a method of obtaining the metal-salen complex derivative.

EFFECT: invention makes it possible to obtain the derivative of a metal-salen complex, characterised by an excellent output and stability.

3 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention represents a pharmaceutical composition for treating demodectic blepharitis and blepharoconjunctivitis, containing recombinant interferon specified in a group: recombinant interferon alpha, recombinant interferon beta, recombinant interferon gamma, boric acid, fluconazole and/or voriconazole with the ingredients of the composition taken in certain ratio, g in 1 ml.

EFFECT: higher therapeutic effect.

2 cl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of obtaining a polymer conjugate of an indolocarbazole compound of formula (I), where R1, R2, R3, W1 and W2 represent hydrogen, X represents methoxy-polyethyleneglycol. The method includes the interaction of a polymer compound of formula (II) with an indolocarbazole compound of formula (III), where Y stands for a methoxygroup. The nvention also relates to a polymer conjugate of formula (I), a pharmaceutical composition, containing the conjugate of formula (I) as an active ingredient, and to the application of the polymer conjugate of formula (I).

EFFECT: obtaining the polymer conjugate of the formula with a high output, the polymer conjugate of the formula for treatment of skin pathologies and HMGB1-associated pathologies.

48 cl, 7 dwg, 7 tbl, 15 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to polypeptides having hydrolysable covalent bonds with therapeutic agents for drug delivery. The invention also refers to pharmaceutical compositions which contain the compounds according to the invention, to using them in methods of treating cancer.

EFFECT: polypeptide conjugates can be used as vectors for a therapeutic agent transport through the blood-brain barrier (BBB) and for delivery into specific types of cells, such as the ovaries, liver, lungs or kidneys.

6 cl, 16 dwg, 8 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine and deals with a pharmaceutical composition for stimulation of growth and reproduction of cells in humans and other animals, which contains suitable pharmaceutical fillers and contains polyethyleneglycol-containing conjugate of the human growth hormone prodrug. The group of inventions also relates to a method of obtaining the said conjugate.

EFFECT: group of inventions provides longer activity of rhGH in comparison with non-modified rhGH, which makes it possible to reduce a dose and a mode of introduction, and reduce a level of lipoatrophy.

13 cl, 27 ex, 11 dwg, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry and represents a composition for treating acne, rosacea and hyperpigmentation, in the form of a gel which contains anchoic acid, a hydrophobic ingredient, a non-aqueous solvent, an emulsifying agent, a gel-forming polymer, a preserving agent, a pH control agent and additionally methylpyrrolidone with anchoic acid having a particle size of less than 100 mcm, while anchoic acid is related to methylpyrrolidone as 1:0.025 to 1:4.

EFFECT: invention provides easy penetration of anchoic acid through a horny layer and its resolution into the oil grand ducts and between skin cells, providing high concentration of anchoic acid that promotes better antibacterial, keratolytic and de-pigmentation effects.

5 cl, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to a conjugate of a version of exendin with a PEG molecule, and can be used in medicine. The above conjugate involves exendin with the amino acid sequence SEQ ID NO: 4 and one PEG molecule with molecular weight 21 kDa to 29 kDa conjugated with a cysteine residue in exendin. The invention also refers to a method for preparing an exendin conjugate, a pharmaceutical composition and a kit for lowering blood glucose providing using the exendin conjugate.

EFFECT: invention enables preparing the exendin conjugate with PEG with GLP-1 receptor agonist activity with maintaining the maximum effect on the stimulation of cAMP (Emax) production in PEGylation.

7 cl, 44 dwg, 5 tbl, 26 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to immunology, and can be used for treating drug abuse, industrial and domestic poisonings, and in man-induced disasters, etc. The invention represents a synthetic immunogen for the protection against toxic action of narcotic and psychoactive substances. The immunogen is presented in the form of a conjugate of a macromolecular carrier specified in: natural or artificial protein, oligo- and polypeptide, carbohydrate, lipid or nucleotide, and haptene - a narcotic or psychotropic compound, and additionally contains poly(4-nitrophenyl)acrylate covalently bond to the conjugate within the range of ratios 2 to 7 moles of the conjugate per one mole of poly(4-nitrophenyl)acrylate with the ratio of haptene and the macromolecular carrier in the conjugate makes 2-17 moles of haptene per 1 mole of the carrier.

EFFECT: using this invention leads to eliciting a stable immune response for a long period of time and providing the body protection against toxic action of the narcotic and psychotropic substances by produced specific haptene antibodies.

3 cl, 4 tbl, 29 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to pharmacology, namely to conjugated proteins, in particular, but not exclusively, to factors of blood coagulation, to methods of obtaining said conjugates, which include stages of interaction of protein or glycoprotein with water-insoluble albumin-binding substance in presence of cyclodextrin molecule, as well as to pharmaceutical compositions, containing said conjugates, and to application of conjugates in manufacturing medication for therapy.

EFFECT: group of inventions ensures increase of bioavailability, period of half-life in vivo, increased resistance to proteases, reduction of immunogenicity.

21 cl, 51 ex

FIELD: medicine.

SUBSTANCE: what is presented is a fused protein that is a Notch1 antagonist, which consists of a human Fc region fused with the EGF-like repeat 1-13 of Notch1 or the EGF-like repeat 1-24 of Notch1. Fc-portion is localised on a carboxy-terminal portion of the EGF-repeat. There are described a pharmaceutical composition for the protein-based Notch signal transmission inhibition and using it for preparing the pharmaceutical composition for treating an individual suffering from: tumour; ovarian cancer; metabolic disorder; vascular proliferative retinopathy. What is presented is using the fused protein for producing the pharmaceutical composition for inhibition: angiogenesis in the individual; physiological lymphangiogenesis or pathological lymphangiogenesis in the individual; tumour deposits in the individual.

EFFECT: using the invention provides the proteins expressed in a supernatant at a level by several times more than the fused protein containing the EGF-like repeats 1-36 of Notch1; they penetrate into the tumour better, maintain a ligand-binding ability with the fused protein containing the repeats 1-24, binds to DLL4 and JAG1, whereas the fused protein containing the repeats 1-13 only binds to DLL4, but not to JAG1 that can find application in therapy of various diseases related to the Notch1 activity.

18 cl, 124 dwg, 10 ex

Up!